U.S. patent application number 15/203103 was filed with the patent office on 2016-10-27 for topk peptides and vaccines including the same.
The applicant listed for this patent is OncoTherapy Science, Inc.. Invention is credited to Yusuke Nakamura, Gaku Nakayama, Ryuji Osawa, Takuya Tsunoda, Tomohisa Watanabe, Sachiko Yoshimura.
Application Number | 20160310586 15/203103 |
Document ID | / |
Family ID | 48167445 |
Filed Date | 2016-10-27 |
United States Patent
Application |
20160310586 |
Kind Code |
A1 |
Nakamura; Yusuke ; et
al. |
October 27, 2016 |
TOPK Peptides and Vaccines Including the Same
Abstract
The present invention provides isolated epitope peptides derived
from TOPK and immunogenic fragments thereof have an ability to
induce cytotoxic T lymphocytes (CTLs) and thus are suitable for use
in cancer immunotherapy, more particularly as cancer vaccines. The
peptides of the present invention encompass both of peptides
including a TOPK-derived amino acid sequence and modified versions
thereof, in which one, two, or several amino acids are substituted,
deleted, inserted and/or added, provided such modified versions
have CTL inducibility. Further provided are polynucleotides
encoding any of the aforementioned peptides as well as
pharmaceutical compositions that include any of the aforementioned
peptides or polynucleotides. The peptides, polynucleotides, and
pharmaceutical compositions of this invention find particular
utility in either or both of the treatment and prevention of a
number of cancers.
Inventors: |
Nakamura; Yusuke; (Tokyo,
JP) ; Tsunoda; Takuya; (Kanagawa, JP) ; Osawa;
Ryuji; (Kanagawa, JP) ; Yoshimura; Sachiko;
(Kanagawa, JP) ; Watanabe; Tomohisa; (Kanagawa,
JP) ; Nakayama; Gaku; (Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OncoTherapy Science, Inc. |
Kanagawa |
|
JP |
|
|
Family ID: |
48167445 |
Appl. No.: |
15/203103 |
Filed: |
July 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14353261 |
Apr 21, 2014 |
9427461 |
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PCT/JP2012/006853 |
Oct 25, 2012 |
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15203103 |
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61552817 |
Oct 28, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/04 20180101;
C12N 9/12 20130101; A61K 39/0011 20130101; A61K 2039/572 20130101;
C07K 14/4748 20130101; A61K 38/00 20130101; A61K 39/001162
20180801; C12N 9/1205 20130101; A61K 39/0005 20130101; A61K
2039/5154 20130101; C07K 16/40 20130101; C07K 2317/34 20130101;
A61P 35/00 20180101; C07K 7/06 20130101; C07K 16/18 20130101; C12Y
207/12002 20130101; G01N 33/505 20130101; G01N 33/574 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; G01N 33/50 20060101 G01N033/50; C12N 9/12 20060101
C12N009/12; C07K 16/40 20060101 C07K016/40 |
Claims
1. An isolated peptide selected from the group consisting of (i)
and (ii) below: (i) an isolated peptide of (a) or (b) below: (a) an
isolated peptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 2, 3, 6, 27 and 28, (b) an
isolated peptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 2, 3, 6, 27 and 28, in which 1,
2, or several amino acid(s) are substituted, inserted, deleted,
and/or added, wherein the peptide has CTL inducibility, (ii) an
isolated peptide of (c) or (d) below: (c) an isolated peptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62, 63, 64,
66, 71, 72 and 76, (d) an isolated peptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 42, 45,
47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and 76, in which 1, 2,
or several amino acid(s) are substituted, inserted, deleted, and/or
added, wherein the peptide has CTL inducibility.
2. The isolated peptide of claim 1, wherein the peptide has one or
both of the following characteristics: (a) the second amino acid
from the N-terminus of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 2, 3, 6, 27 and 28 is substituted
to be an amino acid selected from the group consisting of
phenylalanine, tyrosine, methionine, and tryptophan, and (b) the
C-terminal amino acid of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 2, 3, 6, 27 and 28 is substituted
to be an amino acid selected from the group consisting of
phenylalanine, leucine, isoleucine, tryptophan, and methionine.
3. The isolated peptide of claim 1, wherein the peptide has one or
both of the following characteristics: (a) the second amino acid
from the N-terminus of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62, 63,
64, 66, 71, 72 and 76 is substituted to be an amino acid selected
from the group consisting of leucine and methionine; and (b) the
C-terminal amino acid of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62, 63,
64, 66, 71, 72 and 76 is substituted to be an amino acid selected
from the group consisting of valine and leucine.
4. The isolated peptide of any one of claims 1 to 3, wherein said
peptide has an ability to bind to an HLA antigen.
5. The isolated peptide of claim 4, wherein said HLA antigen is
HLA-A24 or HLA-A2.
6. The isolated peptide of any one of claims 1 to 5, wherein said
peptide is a nonapeptide or a decapeptide.
7. An isolated polynucleotide encoding the isolated peptide of any
one of claims 1 to 6.
8. A composition for inducing a CTL, wherein the composition
comprises one or more peptide(s) of any one of claims 1 to 6, or
one or more polynucleotide(s) of claim 7.
9. A pharmaceutical composition comprising: (a) one or more
peptide(s) of any one of claims 1 to 6, (b) one or more
polynucleotide(s) of claim 7, (c) one or more APC(s) that present a
complex of the peptide of any one of claims 1 to 6 and an HLA
antigen on their surface; (d) one or more exosome(s) that present a
complex of the peptide of any one of claims 1 to 6 and an HLA
antigen on their surface; or (e) one or more CTL(s) that can
recognize a cell presenting a complex of the peptide of any one of
claims 1 to 6 and an HLA antigen on their surface, in combination
with a pharmaceutically acceptable carrier, wherein the
pharmaceutical composition is formulated for the treatment and/or
prophylaxis of cancer, the prevention of a postoperative recurrence
thereof, and/or the induction of an immune response against
cancer.
10. The pharmaceutical composition of claim 9, wherein said
pharmaceutical composition is formulated for administration to a
subject whose HLA antigen is HLA-A24 or HLA-A2.
11. A method for inducing an antigen-presenting cell (APC) with CTL
inducibility, said method comprising the step selected from the
group consisting of: (a) contacting an APC with a peptide of any
one of claims 1 to 6 in vitro, ex vivo or in vivo, and (b)
introducing a polynucleotide encoding the peptide of any one of
claims 1 to 6 into an APC.
12. A method for inducing a CTL, said method comprising a step
selected from the group consisting of: (a) co-culturing a
CD8-positive T cell with an APC that presents on its surface a
complex of an HLA antigen and the peptide of any one of claims 1 to
6, (b) co-culturing a CD8-positive T cells with an exosome that
presents on its surface a complex of an HLA antigen and the peptide
of any one of claims 1 to 6, and (c) introducing into a
CD8-positive T cell a polynucleotide/polynucleotides encoding a T
cell receptor (TCR) subunit polypeptides, wherein the TCR formed by
said TCR subunit polypeptides is capable of binding to a complex of
an HLA antigen and the peptide of any one of claims 1 to 6 on a
cell surface.
13. An isolated APC that presents on its surface a complex of an
HLA antigen and the peptide of any one of claims 1 to 6.
14. The APC of claim 13, which is induced by the method of claim
11.
15. An isolated CTL that targets the peptide of any one of claims 1
to 6.
16. The CTL of claim 15, which is induced by the method of claim
12.
17. A method of inducing an immune response against cancer in a
subject, said method comprising the step of administering to the
subject a composition comprising the peptide of any one of claims 1
to 6, an immunologically active fragment thereof, or a
polynucleotide encoding the peptide or the fragment.
18. An antibody or immunologically active fragment thereof against
the peptide of any one of claims 1 to 6.
19. A vector comprising a nucleotide sequence encoding the peptide
of any one of claims 1 to 6.
20. A host cell transformed or transfected with the vector of claim
19.
21. A diagnostic kit comprising a peptide of any one of claims 1 to
6, the polynucleotide of claim 7 or the antibody of claim 18.
22. A method of screening for a peptide having an ability to induce
a CTL that has specific cytotoxic activity against a cell that
presents a fragment derived from TOPK, wherein the method comprises
the steps of: (i) providing a candidate sequence consisting of an
amino acid sequence modified by substituting, deleting, inserting
and/or adding one, two or several amino acid residues to an
original amino acid sequence, wherein the original amino acid
sequence is selected from the group consisting of SEQ ID NOs: 2, 3,
6, 27, 28, 42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and
76; (ii) selecting a candidate sequence that does not have
substantial significant homology with the peptides derived from any
known human gene products other than TOPK; (iii) contacting a
peptide consisting of the candidate sequence selected in step (ii)
with an antigen presenting cell; (iv) contacting the antigen
presenting cell of step (c) with a CD8-positive T cell; and (v)
identifying the peptide of which CTL inducibility is same to or
higher than a peptide consisting of the original amino acid
sequence.
Description
PRIORITY
[0001] The present application claims the benefit of U.S.
Provisional Applications No. 61/552,817, filed on Oct. 28, 2011,
the entire contents of which are incorporated by reference
herein.
TECHNICAL FIELD
[0002] The present invention relates to the field of biological
science, more specifically to the field of cancer therapy. In
particular, the present invention relates to novel peptides that
are effective as cancer vaccines, and drugs for either or both of
treating and preventing tumors.
BACKGROUND ART
[0003] CD8 positive cytotoxic T lymphocytes (CTLs) have been shown
to recognize epitope peptides derived from tumor-associated
antigens (TAAs) on the major histocompatibility complex (MHC) class
I molecule, and then kill the tumor cells. Since the discovery of
the melanoma antigen (MAGE) family as the first example of TAAs,
many other TAAs have been discovered through immunological
approaches (NPL 1, 2), and some of the TAAs are now in the process
of clinical development as immunotherapeutic targets.
[0004] Favorable TAAs are indispensable for the proliferation and
survival of cancer cells. The use of such TAAs as targets for
immunotherapy may minimize the well-described risk of immune escape
of cancer cells attributable to deletion, mutation, and/or
downregulation of TAAs as a consequence of therapeutically driven
immune selection. Accordingly, the identification of new TAAs
capable of inducing potent and specific anti-tumor immune responses
warrants further development. Thus, clinical application of peptide
vaccination strategies in various types of cancer is ongoing (NPL
3-10). To date, there have been several reports of clinical trials
using these TAAs-derived peptides. Unfortunately, so far, these
cancer vaccine trials have yielded only a low objective response
rate (NPL 11-13). Accordingly, there remains a need in the art for
new TAAs suitable for use as immunotherapeutic targets.
[0005] TOPK (T-LAK cell-originated protein kinase) is a
serine/threonine kinase that is member of the MAPK kinase (MAPKK)
3/6-related MAPKK family. This kinase phosphorylates p38 MAPK and
participate in the regulation of cell cycle check point (NPL 14,
15). Gene expression analysis of TOPK using clinical samples
indicated that TOPK is overexpressed in some malignant cancer, such
as breast cancer, cholangiocarcinoma, hepatocellular carcinoma,
leukemia, colorectal cancer and melanoma (NPL 16-19). Recent
studies indicating that kinase activity plays an important role in
breast carcinogenesis has renewed research interest in
cancer-linked kinases such as TOPK. To that end, Northern blot
analysis has revealed that TOPK transcript is highly expressed in
breast cancer cells but is hardly detectable in normal human
tissues except testis. In addition, knockdown of endogenous TOPK
expression by siRNA in breast cancer cell lines has been shown to
attenuate the cytokinesis and lead to apoptosis of the cancer cells
(NPL 20).
CITATION LIST
Non Patent Literature
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[0007] [NPL 2] Boon T & van der Bruggen P. J Exp Med 1996 Mar.
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Oct. 16, 88(20): 1442-55 [0009] [NPL 4] Butterfield L H et al.,
Cancer Res 1999 Jul. 1, 59(13): 3134-42 [0010] [NPL 5] Vissers J L
et al., Cancer Res 1999 Nov. 1, 59(21): 5554-9 [0011] [NPL 6] van
der Burg S H et al., J Immunol 1996 May 1, 156(9): 3308-14 [0012]
[NPL 7] Tanaka F et al., Cancer Res 1997 Oct. 15, 57(20): 4465-8
[0013] [NPL 8] Fujie T et al., Int J Cancer 1999 Jan. 18, 80(2):
169-72 [0014] [NPL 9] Kikuchi M et al., Int J Cancer 1999 May 5,
81(3): 459-66 [0015] [NPL 10] Oiso M et al., Int J Cancer 1999 May
5, 81(3): 387-94 [0016] [NPL 11] Belli F et al., J Clin Oncol 2002
Oct. 15, 20(20): 4169-80 [0017] [NPL 12] Coulie P G et al., Immunol
Rev 2002 October, 188: 33-42 [0018] [NPL 13] Rosenberg S A et al.,
Nat Med 2004 Sep., 10(9): 909-15 [0019] [NPL 14] Abe Y et. al., J
Bio Chem. 2000 Jul. 14: 21525-21531 [0020] [NPL 15] Ayllon V and
O'connor R., Oncogene. 2007 May 24: 26(24):3451-61 [0021] [NPL 16]
He F et al., Hum Pathol. 2010 March; 41(3):415-24 [0022] [NPL 17]
Li G et al., Ann Hematol. 2006 September; 85(9):583-90 [0023] [NPL
18] Minoo P et al., Int J Oncol. 2010 September; 37(3):707-18
[0024] [NPL 19] Zykova T A et al., Clin Cancer Res. 2006 Dec. 1;
12(23):6884-93 [0025] [NPL 20] Park J H et al., Cancer Res. 2006
Sep. 15; 66(18):9186-95
SUMMARY OF INVENTION
[0026] The present invention is based, at least in part, on the
discovery of novel peptides that may serve as suitable targets of
immunotherapy. Because TAAs are generally perceived by the immune
system as "self" and therefore often have no innate immunogenicity,
the discovery of appropriate targets is of extreme importance.
Through the present invention, TOPK (SEQ ID NO: 86 encoded by the
gene of GenBank Accession No. NM_018492 (SEQ ID NO: 85)) is
demonstrated to be specifically over-expressed in cancer cells, in
particular acute myeloid leukemia (AML), bladder cancer, breast
cancer, cervical cancer, cholangiocellular carcinoma, colorectal
cancer, diffuse-type gastric cancer, non-small cell lung cancer
(NSCLC), lymphoma, osteosarcoma, prostate cancer, renal carcinoma,
small cell lung cancer (SCLC) and soft tissue tumor, but not
limited thereto. Thus, the present invention focuses on TOPK as an
appropriate candidate target of cancer/tumor immunotherapy.
[0027] To that end, the present invention is directed, at least in
part, to the identification of specific epitope peptides among the
gene products of TOPK that possess the ability to induce cytotoxic
T lymphocytes (CTLs) specific to TOPK. As discussed in greater
detail below, peripheral blood mononuclear cells (PBMCs) obtained
from a healthy donor were stimulated using HLA (human leukocyte
antigen)-A*2402 or HLA-A*0201 binding candidate peptides derived
from TOPK. CTL lines were then established with specific
cytotoxicity against the HLA-A24 or HLA-A2 positive target cells
pulsed with each of candidate peptides. The results herein
demonstrate that these peptides are HLA-A24 or HLA-A2 restricted
epitope peptides that can induce potent and specific immune
responses against cells expressing TOPK. These results further
indicate that TOPK is strongly immunogenic and that the epitopes
thereof are effective targets for tumor immunotherapy.
[0028] Accordingly, it is an object of the present invention is to
provide isolated peptides that have an ability to bind an HLA
antigen and include the TOPK sequence (SEQ ID NO: 86) or an
immunogenically active fragment thereof. These peptides are
expected to have CTL inducibility and, thus, can be used to induce
a CTL in vitro, ex vivo or in vivo or to be administered directly
to a subject so as to induce in vivo immune responses against
cancers, examples of which include, but are not limited to, AML,
bladder cancer, breast cancer, cervical cancer, cholangiocellular
carcinoma, colorectal cancer, diffuse-type gastric cancer, NSCLC,
lymphoma, osteosarcoma, prostate cancer, renal carcinoma. SCLC and
soft tissue tumor.
[0029] Preferred peptides are nonapeptides and decapeptides, and
more preferably nonapeptides and decapeptides having an amino acid
sequence selected from among SEQ ID NOs: 2 to 40 and 42 to 84. Of
these, the peptides having an amino sequence selected from among
SEQ ID NOs: 2, 3, 6, 27, 28, 42, 45, 47, 50, 51, 53, 54, 62, 63,
64, 66, 71, 72 and 76 are most preferred.
[0030] The present invention also contemplates modified peptides
having an amino acid sequence selected from among SEQ ID NOs: 2 to
40 and 42 to 84 in which one, two or more amino acids are
substituted, deleted, inserted and/or added, provided the resulting
modified peptides retain the requisite CTL inducibility of the
original unmodified peptide.
[0031] The present invention further encompasses isolated
polynucleotides encoding any one of peptides of the present
invention. These polynucleotides can be used to induce or prepare
antigen-presenting cells (APCs) having CTL inducibility. Like the
above-described peptides of the present invention, such APCs can be
administered to a subject for inducing immune responses against
cancers.
[0032] When administered to a subject, the peptides of the present
invention are preferably presented on the surface of APCs so as to
induce CTLs targeting the respective peptides. Therefore, one
object of the present invention is to provide agents or
compositions for inducing a CTL, such compositions or agents
including one or more peptides of the present invention, or one or
more polynucleotides encoding such peptides. Such agents or
compositions can be used for the treatment and/or prophylaxis of a
primary cancer, a metastasis or post-operative recurrence thereof.
Examples of targeted cancers contemplated by the present invention
include, but are not limited to, AML, bladder cancer, breast
cancer, cervical cancer, cholangiocellular carcinoma, colorectal
cancer, diffuse-type gastric cancer, NSCLC, lymphoma, osteosarcoma,
prostate cancer, renal carcinoma, SCLC and soft tissue tumor.
[0033] The present invention further contemplates pharmaceutical
compositions or agents that include one or more peptides or one or
more polynucleotides of the present invention formulated for the
treatment and/or prophylaxis of a primary cancer, metastasis or
postoperative recurrence cancer as noted above. Instead of or in
addition to the present peptides or polynucleotides, the present
pharmaceutical agents or compositions may include as active
ingredients APCs and/or exosomes that present any of the peptides
of the present invention.
[0034] The peptides or polynucleotides of the present invention may
be used to induce APCs that present on the surface a complex of an
HLA antigen and a peptide of the present invention, for example, by
contacting APCs derived from a subject with the peptide of the
present invention or introducing a polynucleotide encoding the
peptide of the present invention into APCs. Such APCs have high CTL
inducibility against target peptides and are useful for cancer
immunotherapy. Accordingly, the present invention encompasses the
methods for inducing APCs with CTL inducibility as well as the APCs
obtained by the methods.
[0035] It is a further object of the present invention to provide
methods for inducing CTLs, such methods including the step of
co-culturing CD8-positive T cells with APCs presenting on its
surface a complex of an HLA antigen and the peptide of the present
invention, the step of co-culturing CDS8-positive T cells with
exosomes presenting on its surface a complex of an HLA antigen and
the peptide of the present invention, or the step of introducing a
polynucleotide/polynucleotides coding for T cell receptor (TCR)
subunit polypeptides wherein the TCR formed by the subunit
polypeptides can bind to a peptide of the present invention. CTLs
obtained by such methods find use in the treatment and/or
prevention of cancers, more particularly AML, bladder cancer,
breast cancer, cervical cancer, cholangiocellular carcinoma,
colorectal cancer, diffuse-type gastric cancer. NSCLC, lymphoma,
osteosarcoma, prostate cancer, renal carcinoma, SCLC and soft
tissue tumor. Accordingly, the present invention encompasses the
methods for inducing CTLs as well as the CTLs obtained by the
methods. Yet another object of the present invention is to provide
isolated APCs that present on the surface a complex of an HLA
antigen and a peptide of the present invention. The present
invention further provides isolated CTLs that target peptides of
the present invention. These APCs and CTLs may be used for cancer
immunotherapy.
[0036] It is yet another object of the present invention to provide
methods for inducing an immune response against a cancer in a
subject in need thereof, such methods including the step of
administering to the subject a composition that includes at least
one component selected from among a peptide of the present
invention, a polynucleotide encoding such a peptide, exosomes or
APCs presenting such peptides, and CTLs that can recognize cells
presenting such peptides on their surface.
[0037] The applicability of the present invention extends to any of
a number of diseases relating to or arising from TOPK
overexpression, such as cancers expressing TOPK, examples of which
include, but are not limited to, AML, bladder cancer, breast
cancer, cervical cancer, cholangiocellular carcinoma, colorectal
cancer, diffuse-type gastric cancer, NSCLC, lymphoma, osteosarcoma,
prostate cancer, renal carcinoma, SCLC and soft tissue tumor.
[0038] Objects and features of the invention will become more fully
apparent when the following detailed description is read in
conjunction with the accompanying figures and examples. It is to be
understood that both the foregoing summary of the present invention
and the following detailed description are of exemplified
embodiments, and not restrictive of the present invention or other
alternate embodiments of the present invention.
[0039] In particular, while the invention is described herein with
reference to a number of specific embodiments, it will be
appreciated that the description is illustrative of the invention
and is not constructed as limiting of the invention. Various
modifications and applications may occur to those who are skilled
in the art, without departing from the spirit and the scope of the
invention, as described by the appended claims. Likewise, other
objects, features, benefits and advantages of the present invention
will be apparent from this summary and certain embodiments
described below, and will be readily apparent to those skilled in
the art. Such objects, features, benefits and advantages will be
apparent from the above in conjunction with the accompanying
examples, data, figures and all reasonable inferences to be drawn
therefrom, alone or with consideration of the references
incorporated herein.
BRIEF DESCRIPTION OF DRAWINGS
[0040] Various aspects and applications of the present invention
will become apparent to the skilled artisan upon consideration of
the brief description of the figures and the detailed description
of the present invention and its preferred embodiments that
follow.
[0041] FIG. 1 is composed of a series of photographs, (a)-(e),
depicting the results of interferon (IFN)-gamma enzyme-linked
immunospot (ELISPOT) assay on CTLs that were induced with peptides
derived from TOPK. The CTLs in well number #8 induced with
TOPK-A24-9-230 (SEQ ID NO: 2) (a), in #3 induced with
TOPK-A24-9-130 (SEQ ID NO: 3) (b), in #3 induced with
TOPK-A24-9-232 (SEQ ID NO: 6) (c), in #2 induced with
TOPK-A24-10-288 (SEQ ID NO: 27) (d) and in #4 induced with
TOPK-A24-10-289 (SEQ ID NO: 28) (e) showed potent IFN-gamma
production as compared with the control, respectively. The square
on the well of these pictures indicates that the cells from
corresponding well were expanded to establish CTL lines. In
contrast, as is typical of negative data, it no specific IFN-gamma
production was observed from the CTL stimulated with TOPK-A24-9-289
(SEQ ID NO: 1) (f). In the figures, "+" indicates the IFN-gamma
production against target cells pulsed with the appropriate
peptide, and "-" indicates the IFN-gamma production against target
cells not pulsed with any peptides.
[0042] FIG. 2-1 is composed of a series of photographs, (a)-(o),
depicting the results of interferon (IFN)-gamma enzyme-linked
immunospot (ELISPOT) assay on CTLs that were induced with peptides
derived from TOPK. The CTLs in well number #7 induced with
TOPK-A02-9-240 (SEQ ID NO: 42) (a), in #4 induced with
TOPK-A02-9-19 (SEQ ID NO: 45) (b), in #2 induced with
TOPK-A02-9-183 (SEQ ID NO: 47) (c), in #8 induced with
TOPK-A02-9-235 (SEQ ID NO: 50) (d), in #4 induced with
TOPK-A02-9-12 (SEQ ID NO: 51) (e), in #3 induced with
TOPK-A02-9-285 (SEQ ID NO: 53) (f), in #3 induced with
TOPK-A02-9-47 (SEQ ID NO: 54) (g), in #5 induced with
TOPK-A02-10-236 (SEQ ID NO: 62) (h), in #3 induced with
TOPK-A02-10-231 (SEQ ID NO: 63) (i), in #8 induced with
TOPK-A02-10-47 (SEQ ID NO: 64) (j), in #1 induced with
TOPK-A02-10-239 (SEQ ID NO: 66) (k), and in #1 induced with
TOPK-A02-10-272 (SEQ ID NO: 71) (l) showed potent IFN-gamma
production as compared with the control, respectively. The square
on the well of these pictures indicates that the cells from
corresponding well were expanded to establish CTL lines. In
contrast, as is typical of negative data, no specific IFN-gamma
production was observed from the CTL stimulated with TOPK-A02-9-55
(SEQ ID NO: 41) (o). In the figures, "+" indicates the IFN-gamma
production against target cells pulsed with the appropriate
peptide, and "-" indicates the IFN-gamma production against target
cells not pulsed with any peptides.
[0043] FIG. 2-2 is composed of a series of photographs, (a)-(o),
depicting the results of interferon (IFN)-gamma enzyme-linked
immunospot (ELISPOT) assay on CTLs that were induced with peptides
derived from TOPK. The CTLs in #4 induced with TOPK-A02-10-88 (SEQ
ID NO: 72) (m) and in #4 induced with TOPK-A02-10-142 (SEQ ID NO:
76) (n) showed potent IFN-gamma production as compared with the
control, respectively. The square on the well of these pictures
indicates that the cells from corresponding well were expanded to
establish CTL lines. In contrast, as is typical of negative data,
no specific IFN-gamma production was observed from the CTL
stimulated with TOPK-A02-9-55 (SEQ ID NO: 41) (o). In the figures,
"+" indicates the IFN-gamma production against target cells pulsed
with the appropriate peptide, and "-" indicates the IFN-gamma
production against target cells not pulsed with any peptides.
[0044] FIG. 3 is composed of a series of line graphs, (a)-(e),
depicting the IFN-gamma production of the CTL lines stimulated with
TOPK-A24-9-230 (SEQ ID NO: 2) (a), TOPK-A24-9-130 (SEQ ID NO: 3)
(b), TOPK-A24-9-232 (SEQ ID NO: 6) (c), TOPK-A24-10-288 (SEQ ID NO:
27) (d) and TOPK-A24-10-289 (SEQ ID NO: 28) (e). The quantity of
IFN-gamma which CTLs produced was measured by IFN-gamma
enzyme-linked immunosorbent assay (ELISA). The results demonstrate
that CTL lines established by stimulation with each peptide show
potent IFN-gamma production as compared with the control. In the
figures, "+" indicates the IFN-gamma production against target
cells pulsed with the appropriate peptide, and "-" indicates the
IFN-gamma production against target cells not pulsed with any
peptides. R/S ratio indicates the ratio of the number of responder
cells (CTL line) and stimulator cells.
[0045] FIG. 4 is composed of a series of line graphs, (a)-(c),
depicting the IFN-gamma production of the CTL clones established by
limiting dilution from the CTL lines stimulated with TOPK-A24-9-130
(SEQ ID NO: 3) (a), TOPK-A24-10-288 (SEQ ID NO: 27) (b) and
TOPK-A24-10-289 (SEQ ID NO: 28) (c). The results demonstrate that
CTL clones established by stimulation with each peptide show potent
IFN-gamma production as compared with the control. In the figure,
"+" indicates the IFN-gamma production against target cells pulsed
with the appropriate peptide and "-" indicates the IFN-gamma
production against target cells not pulsed with any peptides. R/S
ratio indicates the ratio of the number of responder cells (CTL
clone) and stimulator cells.
[0046] FIG. 5-1 is composed of a series of line graphs, (a)-(f),
depicting the IFN-gamma production of the CTL lines stimulated with
TOPK-A02-9-240 (SEQ ID NO: 42) (a), TOPK-A02-9-19 (SEQ ID NO: 45)
(b), TOPK-A02-9-235 (SEQ ID NO: 50) (c), TOPK-A02-9-12 (SEQ ID NO:
51) (d), TOPK-A02-9-285 (SEQ ID NO: 53) (e), and TOPK-A02-9-47 (SEQ
ID NO: 54) (f). The quantity of IFN-gamma which CTL produced was
measured by IFN-gamma enzyme-linked immunosorbent assay (ELISA).
The results demonstrate that CTL lines established by stimulation
with each peptide show potent IFN-gamma production as compared with
the control. In the figures. "+" indicates the IFN-gamma production
against target cells pulsed with the appropriate peptide, and "-"
indicates the IFN-gamma production against target cells not pulsed
with any peptides. R/S ratio indicates the ratio of the number of
responder cells (CTL line) and stimulator cells.
[0047] FIG. 5-2 is composed of a series of line graphs, (g)-(k),
depicting the IFN-gamma production of the CTL lines stimulated with
TOPK-A02-10-236 (SEQ ID NO: 62) (g), TOPK-A02-10-231 (SEQ ID NO:
63) (h), TOPK-A02-10-47 (SEQ ID NO: 64) (i), TOPK-A02-10-239 (SEQ
ID NO: 66) (j) and TOPK-A02-10-88 (SEQ ID NO: 72) (k). The quantity
of IFN-gamma which CTL produced was measured by IFN-gamma
enzyme-linked immunosorbent assay (ELISA). The results demonstrate
that CTL lines established by stimulation with each peptide show
potent IFN-gamma production as compared with the control. In the
figures. "+" indicates the IFN-gamma production against target
cells pulsed with the appropriate peptide, and "-" indicates the
IFN-gamma production against target cells not pulsed with any
peptides. R/S ratio indicates the ratio of the number of responder
cells (CTL line) and stimulator cells.
[0048] FIG. 6 is composed of a pair of line graphs, (a) and (b),
depicting the IFN-gamma production of the CTL clones established by
limiting dilution from the CTL lines stimulated with TOPK-A02-9-240
(SEQ ID NO: 42) (a) and TOPK-A02-9-285 (SEQ ID NO: 53) (b). The
results demonstrate that the CTL clones established by stimulation
with each peptide show potent IFN-gamma production as compared with
the control. In the figure, "+" indicates the IFN-gamma production
against target cells pulsed with the appropriate peptide and "-"
indicates the IFN-gamma production against target cells not pulsed
with any peptides. R/S ratio indicates the ratio of the number of
responder cells (CTL clone) and stimulator cells.
[0049] FIG. 7 is a line graph depicting the specific CTL activity
of CTL clones against the target cells that express TOPK and
HLA-A*2402. COS7 cells transfected with HLA-A*2402 or the full
length TOPK gene were prepared as the controls. The CT, clone
established with TOPK-A24-10-289 (SEQ ID NO: 28) showed specific
CTL activity against COS7 cells transfected with both TOPK and
HLA-A*2402 (lozenge). On the other hand, no significant specific
CTL activity was detected against target cells expressing either
HLA-A*2402 (triangle) or TOPK (circle).
[0050] FIG. 8 is a line graph depicting the specific CTL activity
of CTL lines against the target cells that express TOPK and
HLA-A*0201. COS7 cells transfected with HLA-A*0201 or the full
length TOPK gene were prepared as the controls. The CTL line
established with TOPK-A02-9-240 (SEQ ID NO: 42) showed specific CTL
activity against COS7 cells transfected with both TOPK and
HLA-A*0201 (lozenge). On the other hand, no significant specific
CTL activity was detected against target cells expressing either
HLA-A*0201 (triangle) or TOPK (circle).
DESCRIPTION OF EMBODIMENTS
[0051] Further to the summary above, it is an object of the present
invention to provide:
[0052] [1] An isolated peptide having CTL inducibility, wherein the
peptide consists of the amino acid sequence of TOPK or an
immunologically active fragment thereof.
[0053] [2] The isolated peptide of [1], wherein the peptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 2, 3, 6, 27, 28, 42, 45, 47, 50, 51, 53, 54, 62, 63,
64, 66, 71, 72 and 76.
[0054] [3] An isolated peptide selected from the group consisting
of (i) and (ii) below:
[0055] (i) an isolated peptide of (a) or (b) below:
[0056] (a) an isolated peptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2, 3, 6, 27 and
28,
[0057] (b) an isolated peptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2, 3, 6, 27 and
28, in which 1, 2, or several amino acid(s) are substituted,
inserted, deleted, and/or added, wherein the peptide has CTL
inducibility,
[0058] (ii) an isolated peptide of (c) or (d) below:
[0059] (c) an isolated peptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 42, 45, 47, 50,
51, 53, 54, 62, 63, 64, 66, 71, 72 and 76,
[0060] (d) an isolated peptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 42, 45, 47, 50,
51, 53, 54, 62, 63, 64, 66, 71, 72 and 76, in which 1, 2, or
several amino acid(s) are substituted, inserted, deleted, and/or
added, wherein the peptide has CTL inducibility.
[0061] [4] The isolated peptide of [3], wherein the peptide has one
or both of the following characteristics:
[0062] (a) the second amino acid from the N-terminus of an amino
acid sequence selected from the group consisting of SEQ ID NOs: 2,
3, 6, 27 and 28 is substituted to be an amino acid selected from
the group consisting of phenylalanine, tyrosine, methionine, and
tryptophan, and
[0063] (b) the C-terminal amino acid of an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2, 3, 6, 27 and
28 is substituted to be an amino acid selected from the group
consisting of phenylalanine, leucine, isoleucine, tryptophan, and
methionine.
[0064] [5] The isolated peptide of [3], wherein the peptide has one
or both of the following characteristics:
[0065] (a) the second amino acid from the N-terminus of an amino
acid sequence selected from the group consisting of SEQ ID NOs: 42,
45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and 76 is
substituted to be an amino acid selected from the group consisting
of leucine and methionine; and
[0066] (b) the C-terminal amino acid of an amino acid sequence
selected from the group consisting of SEQ ID NOs: 42, 45, 47, 50,
51, 53, 54, 62, 63, 64, 66, 71, 72 and 76 is substituted to be an
amino acid selected from the group consisting of valine and
leucine.
[0067] [6] The isolated peptide of any one of [1] to [5], wherein
said peptide has an ability to bind to an HLA antigen.
[0068] [7] The isolated peptide of [6], wherein said HLA antigen is
HLA-A24 or HLA-A2.
[0069] [8] The isolated peptide of any one of [1] to [7], wherein
said peptide is a nonapeptide or a decapeptide.
[0070] [9] An isolated polynucleotide encoding the isolated peptide
of any one of [1] to [8].
[0071] [10] A composition for inducing a CTL, wherein the
composition comprises one or more peptide(s) of any one of [1] to
[8], or one or more polynucleotide(s) of [9].
[0072] [11] A pharmaceutical composition comprising:
(a) one or more peptide(s) of any one of [1] to [8], (b) one or
more polynucleotide(s) of [9], (c) one or more APC(s) that present
a complex of the peptide of any one of [1] to [8] and an HLA
antigen on their surface; (d) one or more exosomes that present a
complex of the peptide of any one of [1] to [8] and an HLA antigen
on their surface or (e) one or more CTLs that can recognize a cell
presenting a complex of the peptide of any one of [1] to [8] and an
HLA antigen on their surface, in combination with a
pharmaceutically acceptable carrier, wherein the pharmaceutical
composition is formulated for the treatment and/or prophylaxis of
cancer, the prevention of a postoperative recurrence thereof,
and/or the induction of an immune response against cancer.
[0073] [12] The pharmaceutical composition of [11], wherein said
pharmaceutical composition is formulated for administration to a
subject whose HLA antigen is HLA-A24 or HLA-A2.
[0074] [13] A method for inducing an antigen-presenting cell (APC)
with CTL inducibility, said method comprising the step selected
from the group consisting of:
(a) contacting an APC with a peptide of any one of [1] to [8] in
vitro, ex vivo or in vivo, and (b) introducing a polynucleotide
encoding the peptide of any one of [1] to [8] into an APC.
[0075] [14] A method for inducing a CTL, said method comprising a
step selected from the group consisting of:
(a) co-culturing a CD8-positive T cell with an APC that presents on
its surface a complex of an HLA antigen and the peptide of any one
of [1] to [8]. (b) co-culturing a CD8-positive T cell with an
exosome that presents on its surface a complex of an HLA antigen
and the peptide of any one of [1] to [8], and (c) introducing into
a CD8-positive T cell a polynucleotide/polynucleotides encoding T
cell receptor (TCR) subunit polypeptides, wherein the TCR formed by
said TCR subunit polypeptides is capable of binding to a complex of
an HLA antigen and the peptide of any one of [11] to [8] on a cell
surface. [15] An isolated APC that presents on its surface a
complex of an HLA antigen and the peptide of any one of [1] to [8].
[16] The APC of [15], which is induced by the method of [13]. [17]
An isolated CTL that targets the peptide of any one of [1] to [8].
[18] The CTL of [17], which is induced by the method of [14]. [19]
A method of inducing an immune response against cancer in a
subject, said method comprising the step of administering to the
subject a composition comprising the peptide of any one of [1] to
[8], an immunologically active fragment thereof, or a
polynucleotide encoding the peptide or the fragment. [20] An
antibody or immunologically active fragment thereof against the
peptide of any one of [1] to [8]. [21]A vector comprising a
nucleotide sequence encoding the peptide of any one of [1] to [8].
[22] A host cell transformed or transfected with the vector of
[21]. [23]A diagnostic kit comprising a peptide of any one of [1]
to [8], the polynucleotide of [9] or the antibody of [20]. [24] A
method of screening for a peptide having an ability to induce a CTL
that has specific cytotoxic activity against a cell that presents a
fragment derived from TOPK, wherein the method comprises the steps
of: (i) providing a candidate sequence consisting of an amino acid
sequence modified by substituting, deleting, inserting and/or
adding one, two or several amino acid residues to an original amino
acid sequence, wherein the original amino acid sequence is selected
from the group consisting of SEQ ID NOs: 2, 3, 6, 27, 28, 42, 45,
47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and 76; (ii) selecting a
candidate sequence that does not have substantial significant
homology with the peptides derived from any known human gene
products other than TOPK: (iii) contacting a peptide consisting of
the candidate sequence selected in step (ii) with an antigen
presenting cell; (iv) contacting the antigen presenting cell of
step (c) with a CD8-positive T cell; and (v) identifying the
peptide of which CTL inducibility is same to or higher than a
peptide consisting of the original amino acid sequence.
[0076] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
embodiments of the present invention, the preferred methods,
devices, and materials are now described. However, before the
present materials and methods are described, it should be
understood that these descriptions are merely illustrative and not
intended to be limited. It should also be understood that the
present invention is not limited to the particular sizes, shapes,
dimensions, materials, methodologies, protocols, etc. described
herein, as these may vary in accordance with routine
experimentation and optimization. Furthermore, the terminology used
in the description is for the purpose of describing the particular
versions or embodiments only, and is not intended to limit the
scope of the present invention which will be limited only by the
appended claims.
[0077] The disclosure of each publication, patent or patent
application mentioned in this specification is specifically
incorporated by reference herein in its entirety. However, nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue or prior
invention.
[0078] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the present invention belongs.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting
I. DEFINITIONS
[0079] The words "a". "an", and "the" as used herein mean "at least
one" unless otherwise specifically indicated.
[0080] The terms "isolated" and "purified" used in relation with a
substance (e.g., peptide, antibody, polynucleotide, etc.) indicates
that the substance is substantially free from at least one
substance that may else be included in the natural source. Thus, an
isolated or purified peptide refers to a peptide that are
substantially free of cellular material such as carbohydrate,
lipid, or other contaminating proteins from the cell or tissue
source from which the peptide is derived, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0081] The term "substantially free of cellular material" includes
preparations of a peptide in which the peptide is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, a peptide that is substantially free
of cellular material includes preparations of polypeptide having
less than about 30%, 20%, 10%, or 5% (by dry weight) of
heterologous protein (also referred to herein as a "contaminating
protein"). When the peptide is recombinantly produced, it is also
preferably substantially free of culture medium, which includes
preparations of peptide with culture medium less than about 20%,
10%, or 5% of the volume of the peptide preparation. When the
peptide is produced by chemical synthesis, it is preferably
substantially free of chemical precursors or other chemicals, which
includes preparations of peptide with chemical precursors or other
chemicals involved in the synthesis of the peptide less than about
30%, 20%, 10%, 5% (by dry weight) of the volume of the peptide
preparation. That a particular peptide preparation contains an
isolated or purified peptide can be shown, for example, by the
appearance of a single band following sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis of the protein preparation
and Coomassie Brilliant Blue staining or the like of the gel. In a
preferred embodiment, peptides and polynucleotides of the present
invention are isolated or purified.
[0082] The terms "polypeptide". "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue(s) is/are one or more modified residue(s),
or non-naturally occurring residue(s), such as an artificial
chemical mimetic of a corresponding naturally occurring amino
acid(s), as well as to naturally occurring amino acid polymers.
[0083] The term "oligopeptide" sometimes used in the present
specification is used to refer to peptides which are 20 amino acid
residues or fewer, typically 15 amino acid residues or fewer in
length and is typically composed of between about 8 and about 11
amino acid residues, often 9 or 10 amino acid residues. The latter
are referred to herein as "nonapeptide" and "decapeptide",
respectively.
[0084] The term "amino acid" as used herein refers to naturally
occurring and synthetic amino acids, as well as amino acid analogs
and amino acid mimetics that similarly function to the naturally
occurring amino acids. Amino acids may be either L-amino acids or
D-amino acids. Naturally occurring amino acids are those encoded by
the genetic code, as well as those modified after translation in
cells (e.g., hydroxyproline, gamma-carboxyglutamate, and
O-phosphoserine). The phrase "amino acid analog" refers to
compounds that have the same basic chemical structure (an alpha
carbon bound to a hydrogen, a carboxy group, an amino group, and an
R group) as a naturally occurring amino acid but have a modified R
group or modified backbones (e.g., homoserine, norleucine,
methionine, sulfoxide, methionine methyl sulfonium). The phrase
"amino acid mimetic" refers to chemical compounds that have
different structures but similar functions to general amino
acids.
[0085] Amino acids may be referred to herein by their commonly
known three letter symbols or the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission.
[0086] The terms "gene", "polynucleotide", "oligonucleotide" and
"nucleic acid" are used interchangeably herein and, unless
otherwise specifically indicated, are referred to by their commonly
accepted single-letter codes.
[0087] The term "agent" and "composition" are used interchangeably
herein to refer to a product that includes specified ingredients in
specified amounts, as well as any product that results, directly or
indirectly, from combination of the specified ingredients in the
specified amounts. Such terms, when used in relation to the
modifier "pharmaceutical" (as in "pharmaceutical agent" and
"pharmaceutical composition") are intended to encompass a product
including the active ingredient(s), and the inert ingredient(s)
that make up the carrier, as well as any product which results,
directly or indirectly, from combination, complexation or
aggregation of any two or more of the ingredients, or from
dissociation of one or more of the ingredients, or from other types
of reactions or interactions of one or more of the ingredients.
Accordingly, in the context of the present invention, the terms
"pharmaceutical agent" and "pharmaceutical composition" refer to
any products made by admixing a molecule or compound of the present
invention and a pharmaceutically or physiologically acceptable
carrier.
[0088] The term "active ingredient" herein refers to a substance in
an agent or composition that is biologically or physiologically
active. Particularly, in the context of a pharmaceutical agent or
composition, the term "active ingredient" refers to a component
substance that shows an objective pharmacological effect. For
example, in case of pharmaceutical agents or compositions for use
in the treatment or prevention of cancer, active ingredients in the
agents or compositions may lead to at least one biological or
physiological action on cancer cells and/or tissues directly or
indirectly. Preferably, such action may include reducing or
inhibiting cancer cell growth, damaging or killing cancer cells
and/or tissues, and so on. Typically, indirect effect of active
ingredients is inductions of CTLs recognizing or killing cancer
cells. Before being formulated, the "active ingredient" may also be
referred to as "bulk". "drug substance" or "technical product".
[0089] The phrase "pharmaceutically acceptable carrier" or
"physiologically acceptable carrier", as used herein, means a
pharmaceutically or physiologically acceptable material,
composition, substance or vehicle, including, but are not limited
to, a liquid or solid filler, diluent, excipient, solvent or
encapsulating material.
[0090] Some pharmaceutical agents or compositions of the present
invention find particular use as vaccines. In the context of the
present invention, the phrase "vaccine" (also referred to as an
"immunogenic composition") refers to an agent or composition that
has the function to improve, enhance and/or induce anti-tumor
immunity upon inoculation into animals.
[0091] Unless otherwise defined, the term "cancer" refers to
cancers or tumors that overexpress the TOPK gene, examples of which
include, but are not limited to, acute myeloid leukemia (AML),
bladder cancer, breast cancer, cervical cancer, cholangiocellular
carcinoma, colorectal cancer, diffuse-type gastric cancer, non
small cell lung cancer (NSCLC), lymphoma, osteosarcoma, prostate
cancer, renal carcinoma, small cell lung cancer (SCLC) and soft
tissue tumor.
[0092] Unless otherwise defined, the terms "cytotoxic T
lymphocyte", "cytotoxic T cell" and "CTL" are used interchangeably
herein and unless otherwise specifically indicated, refer to a
sub-group of T lymphocytes that are capable of recognizing non-self
cells (e.g., tumor/cancer cells, virus-infected cells) and inducing
the death of such cells.
[0093] Unless otherwise defined, the terms "HLA-A24" refers to the
HLA-A24 type containing the subtypes, examples of which include,
but are not limited to, HLA-A*2401, HLA-A*2402, HLA-A*2403,
HLA-A*2404, HLA-A*2407, HLA-A*2408, HLA-A*2420. HLA-A*2425 and
HLA-A*2488.
[0094] Unless otherwise defined, the term "HLA-A2", as used herein,
representatively refers to the subtypes, examples of which include,
but are not limited to, HLA-A*0201, HLA-A*0202, HLA-A*0203,
HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210,
HLA-A*0211, HLA-A*0213, HLA-A*0216, HLA-A*0218, HLA-A*0219,
HLA-A*0228 and HLA-A*0250.
[0095] Unless otherwise defined, the term "kit" as used herein, is
used in reference to a combination of reagents and other materials.
It is contemplated herein that the kit may include microarray,
chip, marker, and so on. It is not intended that the term "kit" be
limited to a particular combination of reagents and/or
materials.
[0096] As used herein, in the context of a subject or patient, the
phrase "subject's (or patient's) HLA antigen is HLA A24 or HLA-A2"
refers to that the subject or patient homozygously or
heterozygously possess HLA-A24 or HLA-A2 antigen gene as an MHC
(major histocompatibility complex) Class I molecule, and HLA-A24 or
HLA-A2 antigen is expressed in cells of the subject or patient as
an HLA antigen.
[0097] To the extent that the methods and compositions of the
present invention find utility in the context of the "treatment" of
cancer, a treatment is deemed "efficacious" if it leads to clinical
benefit such as, reduction in expression of TOPK gene, decrease in
size, prevalence, or metastatic potential of the cancer in a
subject, retarding progression of cancer, alleviation of a clinical
symptom of cancer, prolongation of survival time, suppression of
postoperative recurrence and so on. When the treatment is applied
prophylactically, "efficacious" means that it retards or prevents
cancers from forming or prevents or alleviates a clinical symptom
of cancer. Efficaciousness is determined in association with any
known method for diagnosing or treating the particular tumor
type.
[0098] To the extent that the methods and compositions of the
present invention find utility in the context of the "prevention"
and "prophylaxis" of cancer, such terms are interchangeably used
herein to refer to any activity that reduces the burden of
mortality or morbidity from disease. Prevention and prophylaxis can
occur "at primary, secondary and tertiary prevention levels." While
primary prevention and prophylaxis avoid the development of a
disease, secondary and tertiary levels of prevention and
prophylaxis encompass activities aimed at the prevention and
prophylaxis of the progression of a disease and the emergence of
symptoms as well as reducing the negative impact of an already
established disease by restoring function and reducing
disease-related complications. Alternatively, prevention and
prophylaxis can include a wide range of prophylactic therapies
aimed at alleviating the severity of the particular disorder, e.g.
reducing the proliferation and metastasis of tumors.
[0099] In the context of the present invention, the treatment
and/or prophylaxis of cancer and/or the prevention of postoperative
recurrence thereof include any of the following steps, such as the
surgical removal of cancer cells, the inhibition of the growth of
cancerous cells, the involution or regression of a tumor, the
induction of remission and suppression of occurrence of cancer, the
tumor regression, and the reduction or inhibition of metastasis.
Effective treatment and/or the prophylaxis of cancer decreases
mortality and improves the prognosis of individuals having cancer,
decreases the levels of tumor markers in the blood, and alleviates
detectable symptoms accompanying cancer. For example, reduction or
improvement of symptoms constitutes effectively treating and/or the
prophylaxis include 10%, 20%, 30% or more reduction, or stable
disease.
[0100] In the context of the present invention, the term "antibody"
refers to immunoglobulins and fragments thereof that are
specifically reactive to a designated protein or peptide thereof.
An antibody can include human antibodies, primatized antibodies,
chimeric antibodies, bispecific antibodies, humanized antibodies,
antibodies fused to other proteins or radiolabels, and antibody
fragments. Furthermore, an antibody herein is used in the broadest
sense and specifically covers intact monoclonal antibodies,
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies) formed from at least two intact antibodies, and
antibody fragments so long as they exhibit the desired biological
activity. An "antibody" indicates all classes (e.g., IgA, IgD, IgE,
IgG and IgM).
[0101] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
II. PEPTIDES
[0102] Peptides of the present invention described in detail below
may be referred to as "TOPK peptide(s)" or "TOPK polypeptide(s)".
To demonstrate that peptides derived from TOPK function as an
antigen recognized by CTLs, peptides derived from TOPK (SEQ ID NO:
86) were analyzed to determine whether they were antigen epitopes
restricted by HLA-A24 or HLA-A2 which are commonly encountered HLA
alleles (Date Y et al., Tissue Antigens 47: 93-101, 1996; Kondo A
et al., J Immunol 155: 4307-12, 1995; Kubo R T et al., J Immunol
152: 3913-24, 1994).
[0103] Candidates of HLA-A24 binding peptides derived from TOPK
identified based on their binding affinities to HLA-A24
include:
[0104] TOPK-A24-9-230 (SEQ ID NO: 2), TOPK-A24-9-130 (SEQ ID NO:
3), TOPK-A24-9-237 (SEQ ID NO: 4), TOPK-A24-9-155 (SEQ ID NO: 5),
TOPK-A24-9-232 (SEQ ID NO: 6), TOPK-A24-9-174 (SEQ ID NO: 7),
TOPK-A24-9-73 (SEQ ID NO: 8), TOPK-A24-9-235 (SEQ ID NO: 9),
TOPK-A24-9-19 (SEQ ID NO: 10), TOPK-A24-9-205 (SEQ ID NO: 11),
TOPK-A24-9-77 (SEQ ID NO: 12), TOPK-A24-9-270 (SEQ ID NO: 13),
TOPK-A24-9-58 (SEQ ID NO: 14), TOPK-A24-9-81 (SEQ ID NO: 15),
TOPK-A24-9-278 (SEQ ID NO: 16), TOPK-A24-9-183 (SEQ ID NO: 17),
TOPK-A24-9-227 (SEQ ID NO: 18), TOPK-A24-9-13 (SEQ ID NO: 19),
TOPK-A24-9-146 (SEQ ID NO: 20), TOPK-A24-9-140 (SEQ ID NO: 21),
TOPK-A24-9-103 (SEQ ID NO: 22), TOPK-A24-9-105 (SEQ ID NO: 23),
TOPK-A24-9-118 (SEQ ID NO: 24), TOPK-A24-10-31 (SEQ ID NO: 25),
TOPK-A24-10-155 (SEQ ID NO: 26), TOPK-A24-10-288 (SEQ ID NO: 27),
TOPK-A24-10-289 (SEQ ID NO: 28), TOPK-A24-10-130 (SEQ ID NO: 29),
TOPK-A24-10-47 (SEQ ID NO: 30), TOPK-A24-10-73 (SEQ ID NO: 31),
TOPK-A24-10-102 (SEQ ID NO: 32), TOPK-A24-10-39 (SEQ ID NO: 33),
TOPK-A24-10-4 (SEQ ID NO: 34), TOPK-A24-10-77 (SEQ ID NO: 35),
TOPK-A24-10-241 (SEQ ID NO: 36), TOPK-A24-10-12 (SEQ ID NO: 37),
TOPK-A24-10-148 (SEQ ID NO: 38), TOPK-A24-10-145 (SEQ ID NO: 39)
and TOPK-A24-10-114 (SEQ ID NO: 40).
[0105] Of the above, the following peptides resulted in the
successful establishment of CTLs after in vitro stimulation of
T-cells by dendritic cells (DCs) loaded with these peptides:
[0106] TOPK-A24-9-230 (SEQ ID NO:2), TOPK-A24-9-13) (SEQ ID NO:3),
TOPK-A24-9-232 (SEQ ID NO:6), TOPK-A24-10-288 (SEQ ID NO: 27) and
TOPK-A24-10-289 (SEQ ID NO: 28).
[0107] Candidates of HLA-A2 binding peptides derived from TOPK
identified based on their binding affinities to HLA-A2 include:
[0108] TOPK-A2-9-240 (SEQ ID NO: 42), TOPK-A2-9-34 (SEQ ID NO: 43),
TOPK-A2-9-236 (SEQ ID NO: 44), TOPK-A2-9-19 (SEQ ID NO: 45),
TOPK-A2-9-134 (SEQ ID NO: 46), TOPK-A2-9-183 (SEQ ID NO: 47),
TOPK-A2-9-81 (SEQ ID NO: 48), TOPK-A2-9-149 (SEQ ID NO: 49),
TOPK-A2-9-235 (SEQ ID NO: 50), TOPK-A2-9-12 (SEQ ID NO: 51),
TOPK-A2-9-227 (SEQ ID NO: 52), TOPK-A2-9-285 (SEQ ID NO: 53),
TOPK-A2-9-47 (SEQ ID NO: 54), TOPK-A2-9-310 (SEQ ID NO: 55),
TOPK-A2-9-132 (SEQ ID NO: 56), TOPK-A2-9-242 (SEQ ID NO: 57),
TOPK-A2-9-156 (SEQ ID NO: 58), TOPK-A2-9-138 (SEQ ID NO: 59),
TOPK-A2-9-142 (SEQ ID NO: 60), TOPK-A2-10-190 (SEQ ID NO: 61),
TOPK-A2-10-236 (SEQ ID NO: 62), TOPK-A2-10-231 (SEQ ID NO: 63),
TOPK-A2-10-47 (SEQ ID NO: 64), TOPK-A2-10-234 (SEQ ID NO: 65),
TOPK-A2-10-239 (SEQ ID NO: 66), TOPK-A2-10-290 (SEQ ID NO: 67),
TOPK-A2-10-37 (SEQ ID NO: 68), TOPK-A2-10-20 (SEQ ID NO: 69),
TOPK-A2-10-241 (SEQ ID NO: 70), TOPK-A2-10-272 (SEQ ID NO: 71),
TOPK-A2-10-88 (SEQ ID NO: 72), TOPK-A2-10-81 (SEQ ID NO: 73),
TOPK-A2-10-313 (SEQ ID NO: 74), TOPK-A2-10-54 (SEQ ID NO: 75),
TOPK-A2-10-142 (SEQ ID NO: 76), TOPK-A2-10-35 (SEQ ID NO: 77),
TOPK-A2-10-110 (SEQ ID NO: 78), TOPK-A2-10-223 (SEQ ID NO: 79),
TOPK-A2-10-274 (SEQ ID NO: 80), TOPK-A2-10-173 (SEQ ID NO: 81),
TOPK-A2-10-141 (SEQ ID NO: 82). TOPK-A2-10-292 (SEQ ID NO: 83) and
TOPK-A2-10-180 (SEQ ID NO: 84).
[0109] Of the above, the following peptides resulted in the
successful establishment of CTLs after in vitro stimulation of
T-cells by dendritic cells (DCs) loaded with these peptides:
[0110] TOPK-A02-9-240 (SEQ ID NO:42), TOPK-A02-9-19 (SEQ ID NO:45),
TOPK-A02-9-183 (SEQ ID NO:47), TOPK-A02-9-235 (SEQ ID NO:50),
TOPK-A02-9-12 (SEQ ID NO:51), TOPK-A02-9-285 (SEQ ID NO:53),
TOPK-A02-9-47 (SEQ ID NO:54), TOPK-A02-10-236 (SEQ ID NO:62),
TOPK-A02-10-231 (SEQ ID NO:63), TOPK-A02-10-47 (SEQ ID NO:64),
TOPK-A02-10-239 (SEQ ID NO:66), TOPK-A02-10-272 (SEQ ID NO:71),
TOPK-A02-10-88 (SEQ ID NO:72) and TOPK-A02-10-142 (SEQ ID
NO:76).
[0111] The established CTLs noted above showed potent specific CTL
activity against target cells pulsed with respective peptides.
These results demonstrate that TOPK is an antigen recognized by a
CTL and that the peptides are epitope peptides of TOPK restricted
by HLA-A24 or HLA-A2; therefore, such peptides may be effective as
target antigens for cytotoxicity by CTLs.
[0112] Since the TOPK gene is over-expressed in cancer cells and
tissues, including for example those of AML, bladder cancer, breast
cancer, cervical cancer, cholangiocellular carcinoma, colorectal
cancer, diffuse-type gastric cancer, NSCLC, lymphoma, osteosarcoma,
prostate cancer, renal carcinoma, SCLC and soft tissue tumor, and
not expressed in most normal organs, it represents a good target
for immunotherapy. Thus, the present invention provides
nonapeptides (peptides composed of nine amino acid residues) and
decapeptides (peptides composed of ten amino acid residues)
corresponding to CTL-recognized epitopes from TOPK. Particularly
preferred examples of nonapeptides and decapeptides of the present
invention include those peptides having an amino acid sequence
selected from among SEQ ID NOs: 2 to 40 and 42 to 84.
[0113] Generally, software programs now available, for example, on
the Internet, such as those described in Parker K C et al., J
Immunol 1994, 152(1): 163-75 and Nielsen M et al., Protein Sci
2003; 12: 1007-17 can be used to calculate the binding affinities
between various peptides and HLA antigens in silico. Binding
affinity with HLA antigens can be measured as described, for
example, in Parker K C et al., J Immunol 1994, 152(1): 163-75,
Kuzushima K et al., Blood 2001, 98(6): 1872-81, Larsen M V et al.
BMC Bioinformatics. 2007; 8: 424. Buus S et al. Tissue Antigens,
62:378-84, 2003, Nielsen M et al., Protein Sci 2003; 12: 1007-17,
and Nielsen M et al. PLoS ONE 2007; 2: e796, which are summarized
in, e.g., Lafuente E M et al., Current Pharmaceutical Design, 2009,
15, 3209-3220. Methods for determining binding affinity are
described, for example, in the Journal of Immunological Methods
(1995, 185: 181-190) and Protein Science (2000, 9: 1838-1846).
Therefore, one can readily utilize such software programs to select
those fragments derived from TOPK that have high binding affinity
with HLA antigens using such software programs. Accordingly, the
present invention encompasses peptides composed of any fragments
derived from TOPK, which would be determined to bind with HLA
antigens by such known programs. Furthermore, such peptides may
include the peptide composed of the full length of TOPK
sequence.
[0114] The peptides of the present invention, particularly the
nonapeptides and decapeptides of the present invention, can be
flanked with additional amino acid residues, so long as the
resulting peptide retains its CTL inducibility. The particular
additional amino acid residues can be composed of any kind of amino
acids, so long as they do not impair the CTL inducibility of the
original peptide. Thus, the present invention encompasses peptides
having CTL inducibility, in particular peptides derived from TOPK
(e.g., peptides including an amino acid sequence of SEQ ID NO: 2,
3, 6, 27, 28, 42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72,
or 76). Such peptides are, for example, less than about 40 amino
acids, often less than about 20 amino acids, and usually less than
about 15 amino acids.
[0115] It is generally known that modification of one, two or more
amino acids in a peptide will not influence the function of the
peptide, and in some cases will even enhance the desired function
of the original protein. In fact, modified peptides (i.e., peptides
composed of an amino acid sequence, in which 1, 2 or several amino
acid residues have been modified (i.e., substituted, added, deleted
and/or inserted) as compared to an original reference sequence)
have been known to retain the biological activity of the original
peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6;
Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500;
Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79:
6409-13). Thus, in one embodiment, the peptides of the present
invention have both CTL inducibility and an amino acid sequence
selected from among SEQ ID NOs: 2 to 40 and 42 to 84, in which one,
two or even more amino acids are added, deleted, inserted and/or
substituted. In other words, the peptides of the present invention
have both CTL inducibility and an amino acid sequence in which on,
two or several amino acid(s) are substituted, deleted, inserted
and/or added in the amino acid sequence selected from among SEQ ID
NOs: 2 to 40 and 42 to 84, provided the modified peptides retain
the CTL inducibility of the original peptide.
[0116] Those of skill in the art will recognize that individual
modifications (i.e., deletions, insertions, additions and/or
substitutions) to an amino acid sequence that alter a single amino
acid or a small percentage of the overall amino acid sequence tend
to result in the conservation of the properties of the original
amino acid side-chain. As such, they are often referred to as
"conservative substitutions" or "conservative modifications",
wherein the alteration of a protein results in a modified protein
having a function analogous to the original protein. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Examples of amino acid side-chains
characteristics that are desirable to conserve include, for
example: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and
side-chains having the following functional groups or
characteristics in common: an aliphatic side-chain (G, A, V, L, I,
P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom
containing side-chain (C, M); a carboxylic acid and amide
containing side-chain (D, N, E, Q); a base containing side-chain
(R, K, H); and an aromatic containing side-chain (H, F, Y, W). In
addition, the following eight groups each contain amino acids that
are accepted in the art as conservative substitutions for one
another.
[0117] 1) Alanine (A), Glycine (G);
[0118] 2) Aspartic acid (D), Glutamic acid (E);
[0119] 3) Asparagine (N). Glutamine (Q);
[0120] 4) Arginine (R), Lysine (K);
[0121] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V);
[0122] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0123] 7) Serine (S), Threonine (T); and
[0124] 8) Cysteine (C), Methionine (M) (see, e.g., Creighton,
Proteins 1984).
[0125] Such conservatively modified peptides are also considered to
be peptides of the present invention. However, peptides of the
present invention are not restricted thereto and can include
non-conservative modifications, so long as the resulting modified
peptide retains the CTL inducibility of the original unmodified
peptide. Furthermore, modified peptides should not exclude CTL
inducible peptides derived from polymorphic variants, interspecies
homologues, and alleles of TOPK.
[0126] Amino acid residues may be inserted, substituted and/or
added to the peptides of the present invention or, alternatively,
amino acid residues may be deleted therefrom to achieve a higher
binding affinity. To retain the requisite CTL inducibility, one
preferably modifies (i.e. deletes, inserts, adds and/or
substitutes) only a small number (for example, 1, 2 or several) or
a small percentage of amino acids. Herein, the term "several" means
5 or fewer amino acids, for example, 4 or 3 or fewer. The
percentage of amino acids to be modified is preferably 20% or less,
more preferably 15% or less, and even more preferably 10% or less,
for example 1 to 5%.
[0127] When used in the context of immunotherapy, the peptides of
the present invention should be presented on the surface of a cell
or exosome, preferably as a complex with an HLA antigen. Therefore,
it is preferable to select peptides that not only induce CTLs but
also possess high binding affinity to the HLA antigen. To that end,
the peptides can be modified by substitution, insertion, deletion
and/or addition of the amino acid residues to yield a modified
peptide having improved binding affinity. In addition to peptides
that are naturally displayed, since the regularity of the sequences
of peptides displayed by binding to HLA antigens is already known
(J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; J Immunol
1994, 155: 4307), modifications based on such regularity can be
introduced into the immunogenic peptides of the invention.
[0128] For example, peptides possessing high HLA-A24 binding
affinity tend to have the second amino acid from the N-terminus
substituted with phenylalanine, tyrosine, methionine, or
tryptophan. Likewise, peptides in which the C-terminal amino acid
is substituted with phenylalanine, leucine, isoleucine, tryptophan
or methionine tend to have high HLA-A24 binding affinity.
Accordingly, it may be desirable to substitute the second amino
acid from the N-terminus with phenylalanine, tyrosine, methionine,
or tryptophan, and/or the amino acid at the C-terminus with
phenylalanine, leucine, isoleucine, tryptophan, or methionine in
order to increase the HLA-A24 binding affinity. Thus, peptides
having an amino acid sequence selected from among SEQ ID NOs: 2 to
40 (especially SEQ ID NOs: 2, 3, 6, 27 and 28), in which the second
amino acid from the N-terminus of the amino acid sequence of the
SEQ ID NO is substituted with phenylalanine, tyrosine, methionine,
or tryptophan, and/or in which the C-terminus of the amino acid
sequence of the SEQ ID NO is substituted with phenylalanine,
leucine, isoleucine, tryptophan or methionine are encompassed by
the present invention. Also, the present invention encompasses the
peptides including an amino acid sequence in which one, two or
several amino acid are substituted, deleted, inserted and/or added
in the amino acid sequence selected from among SEQ ID NOs: 2 to 40
(especially SEQ ID NOs: 2, 3, 6, 27 and 28), such peptides having
one or both of the following characteristic of (a) the second amino
acid from the N-terminus is phenylalanine, tyrosine, methionine or
tryptophan; and (b) the C-terminal amino acid is phenylalanine,
leucine, isoleucine, tryptophan or methionine. In preferred
embodiments, the peptides of the present invention include an amino
acid sequence in which the second amino acid from the N-terminus is
substituted with phenylalanine, tyrosine, methionine or tryptophan,
and/or the C-terminal amino acid is substituted with phenylalanine,
leucine, isoleucine, tryptophan or methionine in the amino acid
sequence selected from among SEQ ID NOs: 2 to 40 (especially SEQ ID
NOs: 2, 3, 6, 27 and 28).
[0129] Likewise, peptides showing high HLA-A2 binding affinity tend
to have the second amino acid from the N-terminus substituted with
leucine or methionine and/or the amino acid at the C-terminus
substituted with valine or leucine. Alternatively, it may be
desirable to substitute the second amino acid from the N-terminus
with leucine or methionine, and/or the amino acid at the C-terminus
with valine or leucine in order to increase the HLA-A2 binding
affinity. Thus, peptides having an amino acid sequence selected
from among SEQ ID NOs: 42 to 84 (especially SEQ ID NOs: 42, 45, 47,
50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and 76), in which the second
amino acid from the N-terminus of the amino acid sequence of the
SEQ ID NO is substituted with leucine or methionine, and/or in
which the C-terminus of the amino acid sequence of the SEQ ID NO is
substituted with valine or leucine are encompassed by the present
invention. Also, the present invention encompasses the peptides
including an amino acid sequence in which one, two or several amino
acid are substituted, deleted, inserted and/or added in the amino
acid sequence selected from among SEQ ID NOs: 42 to 84 (especially
SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and
76), such peptides having one or both of the following
characteristic of (a) the second amino acid from the N-terminus is
leucine or methionine; and (b) the C-terminal amino acid is valine
or leucine. In preferred embodiments, the peptides of the present
invention include an amino acid sequence in which the second amino
acid from the N-terminus is substituted with leucine or methionine,
and/or the C-terminal amino acid is substituted with valine or
leucine in the amino acid sequence selected from among SEQ ID NOs:
42 to 84 (especially SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62,
63, 64, 66, 71, 72 and 76).
[0130] Substitutions can be introduced not only at the terminal
amino acids but also at the position of potential T cell receptor
(TCR) recognition of peptides. Several studies have demonstrated
that a peptide with amino acid substitutions can be equal to or
better than the original, for example CAP1, p53.sub.(264-272),
Her-2/neu.sub.(369-377) or gp100.sub.(209-217) (Zaremba et al.
Cancer Res. 57, 4570-4577, 1997, T. K. Hoffmann et al. J Immunol.
(2002); 168(3): 1338-47, S. O. Dionne et al. Cancer Immunol
immunother. (2003) 52: 199-206 and S. O. Dionne et al. Cancer
Immunology, immunotherapy (2004) 53, 307-314).
[0131] The present invention also contemplates the addition of 1, 2
or several amino acids can also be added to the N and/or C-terminus
of the present peptides. Such modified peptides having CTL
inducibility are also included in the present invention.
[0132] For example, the present invention provides an isolated
peptide of less than 15, 14, 13, 12, 11, or 10 amino acids in
length, which has CTL inducibility and comprises the amino acid
sequence selected from the group consisting of:
[0133] (i) an amino acid sequence selected from among SEQ ID NOs: 2
to 24 and 42 to 60,
[0134] (ii) an amino acid sequence in which 1, 2 or several amino
acid(s) are modified in the amino acid sequence selected from the
group consisting of SEQ ID NOs: 2 to 24 and 42 to 60, wherein the
peptide has an ability to induce a cytotoxic T lymphocyte,
[0135] (iii) the amino acid sequence of (ii), wherein, in the
context of HLA-A24, the amino acid sequence has one or both of the
following characteristics:
[0136] (a) the second amino acid frown the N-terminus of said SEQ
ID NOs is or is modified to be an amino acid selected from the
group consisting of phenylalanine, tyrosine, methionine, and
tryptophan, and
[0137] (b) the C-terminal amino acid of said SEQ ID NOs is or is
modified to be an amino acid selected from the group consisting of
phenylalanine, leucine, isoleucine, tryptophan, and methionine,
and
[0138] (iv) the amino acid sequence of (ii), wherein, in the
context of HLA-A2, the amino acid sequence has one or both of the
following characteristics:
[0139] (c) the second amino acid from the N-terminus of said SEQ ID
NO is or is modified to be an amino acid selected from the group
consisting of leucine and methionine; and
[0140] (d) the C-terminal amino acid of said SEQ ID NO is or is
modified to be an amino acid selected from the group consisting of
valine and leucine.
[0141] Moreover, the present invention also provides an isolated
peptide of less than 15, 14, 13, 12, or 11, amino acids in length,
which has CTL inducibility and comprises the amino acid sequence
selected from the group consisting of:
[0142] (i') an amino acid sequence selected form among SEQ ID NOs:
25 to 40 and 61 to 84,
[0143] (ii') an amino acid sequence in which 1, 2 or several amino
acid(s) are modified in the amino acid sequence selected from the
group consisting of SEQ ID NOs: 25 to 40 and 61 to 84, wherein the
peptide has an ability to induce a cytotoxic T lymphocyte,
[0144] (iii') the amino acid sequence of (i'), wherein, in the
context of HLA-A24, the amino acid sequence has one or both of the
following characteristics:
[0145] (a') the second amino acid from the N-terminus of said SEQ
ID NOs is or is modified to be an amino acid selected from the
group consisting of phenylalanine, tyrosine, methionine, and
tryptophan, and
[0146] (b') the C-terminal amino acid of said SEQ ID NOs is or is
modified to be an amino acid selected from the group consisting of
phenylalanine, leucine, isoleucine, tryptophan, and methionine.
[0147] (iii') the amino acid sequence of (i'), wherein, in the
context of HLA-A2, the amino acid sequence has one or both of the
following characteristics:
[0148] (c') the second amino acid from the N-terminus of said SEQ
ID NOs is or is modified to be an amino acid selected from the
group consisting of leucine and methionine; and
[0149] (d') the C-terminal amino acid of said SEQ ID NOs is or is
modified to be an amino acid selected from the group consisting of
valine and leucine.
[0150] These peptides bind with HLA antigens on APCs to be
presented on APCs as complex with an HLA antigen when those
peptides are contacted APCs. Alternatively, those peptides are
introduced into APCs and processed to fragments having an amino
acid sequence selected from among (i)-(iv) and (i')-(iv') in APCs
to be presented on APCs as complexes with HLA antigens, when those
peptides are contacted with APCs. Consequently, CTLs specific to
such peptides are induced.
[0151] However, when the peptide sequence is identical to a portion
of the amino acid sequence of an endogenous or exogenous protein
having a different function, negative side effects such as
autoimmune disorders and/or allergic symptoms against specific
substances may be induced. Therefore, it may be desirable to first
perform homology searches using available databases to avoid
situations in which the sequence of the peptide matches the amino
acid sequence of another protein. When it becomes clear from the
homology searches that no peptide identical to or having 1 or 2
amino acid differences as compared to the objective peptide exists
in nature, the objective peptide can be modified in order to
increase its binding affinity with HLA antigens, and/or increase
its CTL inducibility without any danger of such side effects.
[0152] Although peptides having high binding affinity to the HLA
antigens as described above are expected to be highly effective,
the candidate peptides, which are selected according to the
presence of high binding affinity as an indicator, are further
examined for the presence of CTL inducibility. Herein, the phrase
"CTL inducibility" indicates the ability of the peptide to induce
cytotoxic T lymphocytes (CTLs) when presented on antigen-presenting
cells (APCs). Further, "CTL inducibility" includes the ability of
the peptide to induce CTL activation, CTL proliferation, promote
lysis of target cells by CTL, and to increase IFN-gamma production
by CTL.
[0153] Confirmation of CTL inducibility is accomplished by inducing
APCs carrying human MHC antigens (for example, B-lymphocytes,
macrophages, and dendritic cells (DCs)), or more specifically DCs
derived from human peripheral blood mononuclear leukocytes, and
after stimulation of APCs with a test peptides, mixing APCs with
CD8 positive T cells to induce CTLs, and then measuring the
IFN-gamma produced and released by CTL against the target cells. As
the reaction system, transgenic animals that have been produced to
express a human HLA antigen (for example, those described in
BenMohamed L. Krishnan R. Longmate J, Auge C, Low L, Primus J,
Diamond D J, Hum Immunol 2000, 61(8): 764-79, Related Articles,
Books, Linkout Induction of CTL response by a minimal epitope
vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class
II restricted T(H) response) can be used. Alternatively, the target
cells can be radiolabeled with .sup.51Cr and such, and cytotoxic
activity of CTL can be calculated from radioactivity released from
the target cells. Alternatively, CTL inducibility can be assessed
by measuring IFN-gamma produced and released by CTL in the presence
of APCs that carry immobilized peptides, and visualizing the
inhibition zone on the media using anti-IFN-gamma monoclonal
antibodies.
[0154] As a result of examining the CTL inducibility of the
peptides as described above, it was discovered that nonapeptides or
decapeptides selected from among the amino acid sequences indicated
by SEQ ID NOs: 2, 3, 6, 27, 28, 42, 45, 47, 50, 51, 53, 54, 62, 63,
64, 66, 71, 72 and 76 showed particularly high CTL inducibility as
well as high binding affinity to an HLA antigen. Thus, these
peptides are exemplified as preferred embodiments of the present
invention.
[0155] Furthermore, homology analysis results demonstrated that
such peptides do not have significant homology with peptides
derived from any other known human gene products. Accordingly, the
possibility of unknown or undesired immune responses arising when
used for immunotherapy is lowered. Therefore, also from this
aspect, these peptides are useful for eliciting immunity against
TOPK in cancer patients. Thus, the preferred examples of the
peptides of the present invention include, but are not limited to,
peptides having an amino acid sequence selected from among SEQ ID
NOs: 2, 3, 6, 27, 28, 42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66,
71, 72 and 76 and modified peptides thereof.
[0156] As noted above, the peptides of the present invention has an
ability to induce a CTL specific to TOPK. For example, the peptides
having an amino acid sequence selected from among SEQ ID NOs: 2, 3,
6, 27 and 28, or modified peptides thereof has an ability to induce
a CTL that can show specific cytotoxic activity against a cell
presenting a peptide derived from TOPK via HLA-A24 (e.g., cells
expressing TOPK and HLA-A24). Examples of such cells include
HLA-A24 positive cancer cells. Likewise, the peptides having an
amino acid sequence selected from among SEQ ID NOs: 42, 45, 47, 50,
51, 53, 54, 62, 63, 64, 66, 71, 72 and 76, or modified peptides
thereof has an ability to induce a CTL that can show specific
cytotoxic activity against a cell presenting a peptide derived from
TOPK via HLA-A2 (e.g., cells expressing TOPK and HLA-A2). Examples
of such cells include HLA-A2 positive cancer cells.
[0157] In addition to the above-described modifications, the
peptides of the present invention can also be linked to other
peptides, so long as the resulting linked peptide retains the
requisite CTL inducibility of the original peptide, and more
preferably also retains the requisite HLA binding ability. Examples
of suitable "other" peptides include: the peptides of the present
invention or the CTL-inducible peptides derived from other TAAs.
The peptide of the present invention can be linked "other" peptide
via a linker directly or indirectly. Suitable inter-peptide linkers
are well known in the art and include, for example AAY (P. M.
Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (R. P. M.
Sutmuller et al., J Immunol. 2000, 165: 7308-7315) or K (S. Ota et
al., Can Res. 62, 1471-1476, K. S. Kawamura et al., J Immunol.
2002, 168: 5709-5715).
[0158] For example, non-TOPK tumor associated antigen peptides also
can be used subsequently or simultaneously to increase the immune
response via HLA class I and/or class II. It is well established
that cancer cells can express more than one tumor associated gene.
Thus, it is within the scope of routine experimentation for one of
ordinary skill in the art to determine whether a particular subject
expresses additional tumor associated genes, and then to include
HLA class I-binding peptides and/or HLA class II-binding peptides
derived from such gene products in the pharmaceutical compositions
or vaccines of the present invention.
[0159] Some of HLA class I- and HLA class II-binding peptides are
known to those of ordinary skill in the art (for example, see
Coulie, Stem Cells 13:393-403, 1995), and can be used in the
present invention in a like manner as those disclosed herein. Thus,
one of ordinary skill in the art can readily prepare polypeptides
including one or more TOPK peptides and one or more of the non-TOPK
peptides, or nucleic acids encoding such polypeptides, using
standard procedures of molecular biology.
[0160] The above described linked peptides are referred to herein
as "polytopes", i.e., groups of two or more potentially immunogenic
or immune response stimulating peptides which can be joined
together in various arrangements (e.g., concatenated, overlapping).
The polytope (or nucleic acid encoding the polytope) can be
administered in a standard immunization protocol. e.g., to animals,
to test the effectiveness of the polytope in stimulating, enhancing
and/or provoking an immune response.
[0161] The peptides can be joined together directly or via the use
of flanking sequences to form polytopes, and the use of polytopes
as vaccines is well known in the art (see. e.g., Thomson et al.,
Proc. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al.,
Nature Biotechnol. 15(12):1280-1284, 1997; Thomson et al., J
Immunol. 157(2):822-826, 1996; Tam et al., J Exp. Med.
171(1):299-306, 1990). Polytopes containing various numbers and
combinations of epitopes can be prepared and tested for recognition
by CTLs and for efficacy in increasing an immune response.
[0162] The peptides of the present invention can also be linked to
other substances, so long as the resulting linked peptide retains
the requisite CTL inducibility of the original peptide. Examples of
suitable substances include, for example: peptides, lipids, sugar
and sugar chains, acetyl groups, natural and synthetic polymers,
etc. The peptides can contain modifications such as glycosylation,
side chain oxidation, or phosphorylation, etc., provided the
modifications do not destroy the biological activity of the
original peptide. These kinds of modifications can be performed to
confer additional functions (e.g., targeting function, and delivery
function) or to stabilize the peptide.
[0163] For example, to increase the in vive stability of a peptide,
it is known in the art to introduce D-amino acids, amino acid
mimetics or unnatural amino acids; this concept can also be adapted
to the present peptides. The stability of a peptide can be assayed
in a number of ways. For instance, peptidases and various
biological media, such as human plasma and serum, can be used to
test stability (see, e.g., Verhoef et al., Eur J Drug Metab
Pharmacokin 1986, 11: 291-302).
[0164] Moreover, as noted above, among the modified peptides that
are substituted, deleted inserted or added by 1, 2 or several amino
acid residues, those having same or higher activity as compared to
original peptides can be screened for or selected. The present
invention, therefore, also provides the method of screening for or
selecting modified peptides having same or higher activity as
compared to originals. An illustrative method includes the steps
of:
[0165] a: modifying (i.e., substituting, deleting, inserting or
adding) at least one amino acid residue of a peptide of the present
invention,
[0166] b: determining the activity of the peptide,
[0167] c: selecting the peptide having same or higher activity as
compared to the original.
[0168] In preferred embodiments, the present invention provides a
method of screening for a peptide having an ability to induce a CTL
that has specific cytotoxic activity against a cell that presents a
fragment derived from TOPK, wherein the method comprises the steps
of:
[0169] (i) providing a candidate sequence consisting of an amino
acid sequence modified by substituting, deleting, inserting and/or
adding one, two or several amino acid residues to an original amino
acid sequence, wherein the original amino acid sequence is selected
from the group consisting of SEQ ID NOs: 2, 3, 6, 27, 28, 42, 45,
47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and 76;
[0170] (ii) selecting a candidate sequence that does not have
substantial significant homology (or sequence identity) with the
peptides derived from any known human gene products other than
TOPK:
[0171] (iii) contacting a peptide consisting of the candidate
sequence selected in step (ii) with an antigen presenting cell;
[0172] (iv) contacting the antigen presenting cell of step (c) with
a CD8 positive T cell; and
[0173] (v) identifying the peptide of which CTL inducibility is
same to or higher than a peptide consisting of the original amino
acid sequence.
Herein, the activity to be assayed may include MHC binding
activity, APC or CTL inducibility and cytotoxic activity.
Preferably, the activity of the peptide to be assayed is CTL
inducibility.
III. PREPARATION OF TOPK PEPTIDES
[0174] The peptides of the present invention can be prepared using
well known techniques. For example, the peptides can be prepared
synthetically, using recombinant DNA technology or chemical
synthesis. The peptides of the present invention can be synthesized
individually or as longer polypeptides including two or more
peptides. The peptides can then be isolated i.e., purified or
isolated so as to be substantially free of other naturally
occurring host cell proteins and fragments thereof, or any other
chemical substances.
[0175] The peptides of the present invention may contain
modifications, such as glycosylation, side chain oxidation, or
phosphorylation, provided the modifications do not destroy the
biological activity of the original peptide. Other illustrative
modifications include incorporation of one or more D-amino acids or
other amino acid mimetics that can be used, for example, to
increase the serum half life of the peptides.
[0176] Peptides of the present invention can be obtained through
chemical synthesis based on the selected amino acid sequence.
Examples of conventional peptide synthesis methods that can be
adapted for the synthesis include:
[0177] (i) Peptide Synthesis, Interscience, New York, 1966;
[0178] (ii) The Proteins, Vol. 2, Academic Press, New York,
1976;
[0179] (iii) Peptide Synthesis (in Japanese), Maruzen Co.,
1975:
[0180] (iv) Basics and Experiment of Peptide Synthesis (in
Japanese), Maruzen Co., 1985;
[0181] (v) Development of Pharmaceuticals (second volume) (in
Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
[0182] (vi) WO99/67288; and
[0183] (vii) Barany G. & Merrifield R. B., Peptides Vol. 2.
"Solid Phase Peptide Synthesis", Academic Press, New York, 1980,
100-118.
[0184] Alternatively, the present peptides can be obtained adapting
any known genetic engineering method for producing peptides (e.g.,
Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss &
Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
For example, first, a suitable vector harboring a polynucleotide
encoding the objective peptide in an expressible form (e.g.,
downstream of a regulatory sequence corresponding to a promoter
sequence) is prepared and transformed into a suitable host cell.
The host cell is then cultured to produce the peptide of interest.
The peptide can also be produced in vitro adopting an in vitro
translation system.
IV. POLYNUCLEOTIDES
[0185] The present invention also provides a polynucleotide that
encodes any of the aforementioned peptides of the present
invention. These include polynucleotides derived from the natural
occurring TOPK gene (e.g., GenBank Accession No. NM_018492 (SEQ ID
NO: 85)) as well as those having a conservatively modified
nucleotide sequence thereof. Herein, the phrase "conservatively
modified nucleotide sequence" refers to sequences which encode
identical or essentially identical amino acid sequences. Due to the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a peptide also describes every possible silent
variation of the nucleic acid. One of ordinary skill in the art
will recognize that each codon in a nucleic acid (except AUG, which
is ordinarily the only codon for methionine, and TGG, which is
ordinarily the only codon for tryptophan) can be modified to yield
a functionally identical molecule. Accordingly, each silent
variation of a nucleic acid that encodes a peptide is implicitly
described in each disclosed sequence.
[0186] The polynucleotide of the present invention can be composed
of DNA, RNA, and derivatives thereof. As is well known in the art,
a DNA is suitably composed of bases such as A, T, C, and G, and T
is replaced by U in an RNA. One of skill will recognize that
non-naturally occurring bases may be included in polynucleotides,
as well.
[0187] The polynucleotide of the present invention can encode
multiple peptides of the present invention with or without
intervening amino acid sequences in between. For example, the
intervening amino acid sequence can provide a cleavage site (e.g.,
enzyme recognition sequence) of the polynucleotide or the
translated peptides. Furthermore, the polynucleotide can include
any additional sequences to the coding sequence encoding the
peptide of the present invention. For example, the polynucleotide
can be a recombinant polynucleotide that includes regulatory
sequences required for the expression of the peptide or can be an
expression vector (plasmid) with marker genes and such. In general,
such recombinant polynucleotides can be prepared by the
manipulation of polynucleotides through conventional recombinant
techniques using, for example, polymerases and endonucleases.
[0188] Both recombinant and chemical synthesis techniques can be
used to produce the polynucleotides of the present invention. For
example, a polynucleotide can be produced by insertion into an
appropriate vector, which can be expressed when transfected into a
competent cell. Alternatively, a polynucleotide can be amplified
using PCR techniques or expression in suitable hosts (see, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1989). Alternatively, a
polynucleotide can be synthesized using the solid phase techniques,
as described in Beaucage S L & Iyer R P, Tetrahedron 1992, 48:
2223-311: Matthes et al., EMBO J 1984, 3: 801-5.
V. EXOSOMES
[0189] The present invention further provides intracellular
vesicles, called exosomes, that present complexes formed between
the peptides of the present invention and HLA antigens on their
surface. Exosomes can be prepared, for example, using the methods
detailed in Japanese Patent Application Kohyo Publications Nos. Hei
11-510507 and WO99/03499, and can be prepared using APCs obtained
from patients who are subject to treatment and/or prevention. The
exosomes of the present invention can be inoculated as vaccines, in
a fashion similar to the peptides of the present invention.
[0190] The type of HLA antigens included in the complexes must
match that of the subject requiring treatment and/or prevention.
For example, in the Japanese population. HLA-A24 and HLA-A2,
particularly HLA-A*2402 and HLA-A*0201 and HLA-A*0206, are
prevalent and therefore would be appropriate for treatment of
Japanese patients. The use of the HLA-A24 type that are highly
expressed among the Japanese and Caucasian is favorable for
obtaining effective results, and subtypes such as HLA-A*2402,
HLA-A*0201 and HLA-A*0206 also find use. Typically, in the clinic,
the type of HLA antigen of the patient requiring treatment is
investigated in advance, which enables the appropriate selection of
peptides having high levels of binding affinity to the particular
antigen, or having CTL inducibility by antigen presentation.
Furthermore, in order to obtain peptides having both high binding
affinity and CTL inducibility, substitution, insertion, deletion
and/or addition of 1, 2, or several amino acids can be performed
based on the amino acid sequence of the naturally occurring TOPK
partial peptide.
[0191] When the exosome of the present invention possess HLA-A24
type as an antigen, the peptides including the amino acid sequence
selected from among SEQ ID NOs: 2 to 40 (especially SEQ ID NOs: 2,
3, 6, 27 and 28) have particular utility.
[0192] Alternatively, when the exosome of the present invention
possess HLA-A2 type as an antigen, the peptides including the amino
acid sequence selected from among SEQ ID NOs: 42 to 84 (especially
SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 and
76) have particular utility.
[0193] In some embodiments, the exosomes of the present invention
are exosomes that present a complex of the peptide of the present
invention and HLA-A24 or HLA-A2 antigen on their surface. In
typical embodiments, the exosome of the presents invention present
a complex of a peptide having an amino acid sequence of SEQ ID NO:
2, 3, 6, 27 or 28 (or modified peptide thereof) and HLA-A24 on its
surface. In other embodiments, the exosome of the present invention
presents a complex of a peptide having an amino acid sequence of
SEQ ID NO: 42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 or 76
(or modified peptide thereof) and HLA-A2 on its surface.
VI. ANTIGEN-PRESENTING CELLS (APCS)
[0194] The present invention also provides isolated
antigen-presenting cells (APCs) that present complexes formed
between HLA antigens and the peptides of the present invention on
its surface. The APCs can be derived from patients who are subject
to treatment and/or prevention, and can be administered as vaccines
by themselves or in combination with other drugs including the
peptides, exosomes, or CTLs of the present invention.
[0195] The APCs are not limited to a particular kind of cells.
Examples of APCs include, but are not limited to, dendritic cells
(DCs). Langerhans cells, macrophages, B cells, and activated T
cells, which are known to present proteinaceous antigens on their
cell surface so as to be recognized by lymphocytes. Since DCs are
representative APCs having the strongest CTL inducing activity
among APCs, DCs can be preferably used as the APCs of the present
invention.
[0196] For example, the APCs of the present invention can be
obtained by inducing DCs from peripheral blood monocytes and then
contacting (stimulating) them with the peptides of the present
invention in vitro, ex vivo or in vivo. When the peptides of the
present invention are administered to the subjects, APCs that
present the peptides of the present invention are induced in the
body of the subject. Herein, the phrase "inducing an APC" includes
contacting (stimulating) an antigen-presenting cell with the
peptides of the present invention, or introducing a polynucleotide
encoding the peptide of the present invention into an
antigen-presenting cell to have the APC present a complex formed
between an HLA antigen and a peptide of the present invention on
its surface. For example, the APCs of the present invention can be
obtained by collecting APCs from a subject after administering one
or more peptides of the present invention to the subject.
Alternatively, the APCs of the present invention can be obtained by
contacting APCs, which have been collected from a subject, with the
peptide of the present invention.
[0197] The APCs of the present invention can be administered to a
subject for inducing immune response against cancer in the subject
by themselves or in combination with other drugs including the
peptides, exosomes or CTLs of the present invention. For example,
the ex vivo administration can include steps of:
[0198] a: collecting APCs from a first subject.
[0199] b: contacting the APCs of step a, with the peptide, and
[0200] c: administering the APCs of step b to a second subject.
[0201] The first subject and the second subject can be the same
individual, or may be different individuals. The APCs obtained by
step b can be formulated and administered a vaccine for treating
and/or preventing cancer, such as bladder cancer, breast cancer,
cervical cancer, cholangiocellular carcinoma, CML, colorectal
cancer, esophageal cancer, gastric cancer, diffuse-type gastric
cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic
cancer, prostate cancer. SCLC, soft tissue tumor and testicular
tumor, but not limited thereto.
[0202] In the context of the present invention, one may utilize one
or more peptides of the present invention for manufacturing a
pharmaceutical composition for inducing an antigen-presenting cell.
A method or process for manufacturing a pharmaceutical composition
for inducing an antigen-presenting cell is provided herein and
preferably includes the step of admixing or formulating the peptide
of the invention with a pharmaceutically acceptable carrier.
[0203] The present invention also provides for the use of the
peptide of the present invention for inducing an antigen-presenting
cell.
[0204] According to an aspect of the present invention, the APCs of
the present invention have CTL inducibility. In the context of the
APCs, the phrase "CTL inducibility" refers to the ability of an APC
to induce a CTL when contacted with a CD8 positive T cell. Further,
"CTL inducibility" includes the ability of an APC to induce CTL
activation, CTL proliferation, promote lysis of a target cell by a
CTL, and to increase IFN-gamma production by a CTL. In particular,
the APCs of the present invention have an ability to induce CTLs
specific to TOPK.
[0205] Such APCs having CTL inducibility can be prepared by a
method that includes the step of transferring a polynucleotide
encoding the peptide of the present invention to APCs in vitro as
well as the method mentioned above. The introduced polynucleotide
can be in the form of DNAs or RNAs. Examples of methods for
introduction include, without particular limitations, various
methods conventionally performed in this field, such as
lipofection, electroporation, and calcium phosphate method can be
used. More specifically, it can be performed as described in Cancer
Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996,
184: 465-72; Published Japanese Translation of International
Publication No. 2000-509281. By transferring the gene encoding the
peptide of the present invention into APCs, the gene undergoes
transcription, translation, and such in the cell, and then the
obtained protein is processed by MHC Class I or Class II, and
proceeds through a presentation pathway to present the peptides of
the present invention. Alternatively, the APCs of the present
invention can be prepared by a method which induces the step of
contacting APCs with the peptide of the present invention.
[0206] In some embodiments, the APCs of the present invention are
APCs that present complexes of HLA-A24 or HLA-A2 antigen and the
peptide of the present invention on their surface. In typical
embodiments, the APC of the present invention presents a complex of
a peptide having an amino acid sequence of SEQ ID NO: 2, 3, 6, 27
or 28 (or modified peptide thereof) and HLA-A24 on its surface. In
other embodiments, the APC of the present invention presents a
complex of a peptide having an amino acid sequence of SEQ ID NO:
42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 or 76 (or
modified peptide thereof) and HLA-A2 on its surface.
VII. CYTOTOXIC T LYMPHOCYTES (CTLS)
[0207] A CTL induced against any one of the peptides of the present
invention strengthens the immune response targeting cancer cells in
vivo and thus can be used as vaccines, in a fashion similar to the
peptides per se. Thus, the present invention provides isolated CTLs
that are specifically induced or activated by any one of the
peptides of the present invention.
[0208] Such CTLs can be obtained by (1) administering the
peptide(s) of the present invention to a subject, (2) contacting
(stimulating) subject-derived APCs, and CD8-positive T cells, or
peripheral blood mononuclear leukocytes in vitro with the
peptide(s) of the present invention, (3) contacting CD8-positive T
cells or peripheral blood mononuclear leukocytes in vitro with the
APCs or exosomes presenting a complex of an HLA antigen and the
peptide of the present invention on its surface, or (4) introducing
a polynucleotide/polynucleotides encoding T cell receptor (TCR)
subunits that can form a TCR having an ability to bind to a complex
of an HLA antigen and the peptide of the present invention on a
cell surface. Such APCs or exosomes for the method of (3) can be
prepared by the methods described above. Details of the method of
(4) is described bellow in section "VIII. T Cell Receptor
(TCR)".
[0209] The CTLs of the present invention can be derived from
patients who are subject to treatment and/or prevention, and can be
administered by themselves or in combination with other drugs
including the peptides, APC or exosomes for the purpose of
regulating effects. The obtained CTLs act specifically against
target cells presenting the peptides of the present invention, for
example, the same peptides used for induction. The target cells can
be cells that endogenously express TOPK, such as cancer cells, or
cells that are transfected with the TOPK gene; and cells that
present a peptide of the present invention on the cell surface due
to stimulation by the peptide can also serve as targets of
activated CTL attack.
[0210] In some embodiments, the CTLs of the present invention can
recognize cells presenting complexes of an HLA-A24 or HLA-A2
antigen and the peptide of the present invention on their surface.
In the context of CTLs, the phrase "recognize a cell" refers to
binding a complex of an HLA-A24 or HLA-A2 antigen and the peptide
of the present invention on the cell surface via its TCR and
showing specific cytotoxic activity against the cell. Herein,
"specific cytotoxic activity" refers to showing cytotoxic activity
against the cell presenting a complex of an HLA-A24 or HLA-A2
antigen and the peptide of the present invention but not other
cells. Accordingly, the CTLs that show specific cytotoxic activity
against a cell presenting the peptide of the present invention are
included in the present invention.
[0211] In typical embodiments, the CTL of the present invention can
recognize a cell presenting a peptide having an amino acid sequence
of SEQ ID NO: 2, 3, 6, 27 or 28 (or modified peptide thereof) via
an HLA-A24. In preferred embodiments, such CTL of the present
invention can recognize a cell expressing TOPK and an HLA-A24
(e.g., HLA-A24 positive cancer cell).
[0212] In other embodiments, the CTL of the present invention can
recognize a cell presenting a peptide having an amino acid sequence
of SEQ ID NO: 42, 45, 47, 50, 51, 53, 54, 62, 63, 64, 66, 71, 72 or
76 (or modified peptide thereof) via an HLA A2. In preferred
embodiments, such CTL of the present invention can recognize a cell
expressing TOPK and an HLA-A2 (e.g., HLA-A2 positive cancer
cell).
VII. T CELL RECEPTOR (TCR)
[0213] The present invention also provides a composition that
includes one or more polynucleotides encoding polypeptides that are
capable of forming a subunit of a T cell receptor (TCR), and
methods of using the same. Such TCR subunits have the ability to
form TCRs that confer specificity to T cells against tumor cells
expressing TOPK. By using known methods in the art, the
polynucleotide encoding each of alpha- and beta-chains as the TCR
subunits of the CTL induced with the peptides of the present
invention can be identified (WO2007/032255 and Morgan et al., J
Immunol, 171, 3288 (2003)). For example, the PCR method is
preferred to analyze the TCR. The PCR primers for the analysis can
be, for example, 5'-R primers (5'-gtctaccaggcattcgcttcat-3') (SEQ
ID NO: 87) as a 5' side primer, and 3-TRa-C primers
(5'-tcagctggaccaccacagccgcagcgt-3') (SEQ ID NO: 88) specific to TCR
alpha chain C region, 3-TRb-C1 primers
(5'-tcagaaatcctttctcttgac-3') (SEQ ID NO: 89) specific to TCR beta
chain C1 region or 3-TRbeta-C2 primers
(5'-ctagctctggaatcctttctctt-3') (SEQ ID NO: 90) specific to TCR
beta chain C2 region as 3' side primers, but not limited thereto.
The derivative TCRs can bind target cells presenting the TOPK
peptide with high avidity, and optionally mediate efficient killing
of target cells presenting the TOPK peptide of the present
invention in vivo and in vitro.
[0214] The polynucleotide/polynucleotides encoding the TCR subunits
(i.e., the polynucleotide encoding both of the TCR subunits or
polynucleotides encoding each of TCR subunits) can be incorporated
into suitable vectors, e.g., retroviral vectors. These vectors are
well known in the art. The polynucleotides or the vectors including
them usefully can be transferred into a T cell (e.g., CD8-positive
T cell), for example, a T cell from a patient. Advantageously, the
present invention provides an off-the-shelf composition allowing
rapid modification of a patient's own T cells (or those of another
mammal) to rapidly and easily produce modified T cells having
excellent cancer cell killing properties.
[0215] Specific TCRs against the peptides of the present invention
should be capable of specifically recognizing a complex of a
peptide of the present invention and an HLA antigen, giving a T
cell specific activity against the target cell presenting a complex
of the peptide of the present invention and an HLA antigen when the
TCR is expressed on the surface of the T cell. The requisite
activity can be confirmed by any known methods that CTL prepared by
introducing the polypeptide(s) encoding such TCR subunits can be
specifically recognize such target cells. Preferred examples of
such method include, for example, tetramer analysis using HLA
molecules and the peptides of the present invention, and ELISPOT
assay. By ELISPOT assay, it can be confirmed that CTLs prepared by
the method as describe above can specifically recognize the target
cells, and that the signals generated by such recognition by
transmitted intracellularly. Furthermore, it can be confirmed by a
known method that CTLs prepared by the method described above have
specific cytotoxic activity against the target cells. Examples of
such methods includes, for example, chromium release assay using
cells expressing both of TOPK and HLA-A24 or HLA-A2.
[0216] In one aspect, the present invention provides CTLs that are
prepared by transduction with the polypeptide/polypeptides encoding
the TCR subunit polypeptides (i.e., the polynucleotide encoding
both of the TCR subunits or polynucleotides encoding each of TCR
subunits), wherein the TCR formed by such TCR subunits can bind to
a complex of the TOPK peptide having an amino acid sequence
selected from among SEQ ID) NOs: 2 to 40 and an HLA-A24 antigen on
cell surface, or can bind to a complex of the TOPK peptide having
an amino acid sequence selected from among SEQ ID NOs: 42 to 84 and
an HLA-A2 antigen on cell surface.
[0217] The transduced CTLs are capable of homing to cancer cells in
vivo, and can be expanded by well known culturing methods in vitro
(e.g., Kawakami et al., J Immunol., 142, 3452-3461 (1989)). The
CTLs of the present invention can be used to form an immunogenic
composition useful in either or both of the treatment and the
prevention of cancer in a patient in need of therapy or protection
(See WO200631221 the contents of which are incorporated by
reference herein).
IX. PHARMACEUTICAL AGENTS OR COMPOSITIONS
[0218] Since TOPK expression is specifically elevated in cancers,
examples of which include, but are not necessarily limited to, AML,
bladder cancer, breast cancer, cervical cancer, cholangiocellular
carcinoma, colorectal cancer, diffuse-type gastric cancer. NSCLC,
lymphoma, osteosarcoma, prostate cancer, renal carcinoma, SCLC and
soft tissue tumor as compared with normal tissue, the peptides or
polynucleotides of the present invention may be used to induce an
immune response against cancer and thus serve to treat and/or
prevent cancer and/or to prevent a metastatic or postoperative
recurrence thereof. Thus, the present invention provides
pharmaceutical compositions or agents formulated for the treatment
and/or prophylaxis of cancer, and/or for the prevention of a
postoperative recurrence thereof, such compositions or agents
including one or more of the peptides, or polynucleotides of the
present invention as one or more active ingredients. Alternatively,
the peptides of the present invention can be expressed on the
surface of any of the foregoing exosomes or cells, such as APCs,
for the use as pharmaceutical compositions or agents. In addition,
the aforementioned CTLs which target any one of the peptides of the
present invention can also be used as the active ingredient of the
present pharmaceutical compositions or agents.
[0219] Accordingly, the present invention provides agents or
compositions including at least one active ingredient selected from
among:
[0220] (a) one or more peptides of the present invention;
[0221] (b) one or more polynucleotides encoding such a peptide of
the present invention in an expressible form;
[0222] (c) one or more APCs or an exosomes of the present
invention; and
[0223] (d) one or more CTLs of the present invention.
[0224] The pharmaceutical compositions or agents of the present
invention also find use as a vaccine. In the context of the present
invention, the phrase "vaccine" (also referred to as an
"immunogenic composition") refers to an agent or composition that
has the function to improve, enhance and/or induce anti-tumor
immunity upon inoculation into an animal. In other words, the
present invention provides the pharmaceutical agents or
compositions for inducing an immune response against cancer in a
subject.
[0225] The pharmaceutical compositions or agents of the present
invention can be used to treat and/or prevent cancer and/or prevent
a postoperative or metastatic recurrence thereof in subjects or
patients. Examples of such subjects include humans as well as other
mammals including, but not limited to, mice, rats, guinea-pigs,
rabbits, cats, dogs, sheep, goats, pigs, cattle, horses, monkeys,
baboons, and chimpanzees, particularly commercially important
animals or domesticated animals. In some embodiments, the
pharmaceutical agents or compositions of the present invention can
be formulated for the administration to a subject whose HLA antigen
is HLA-A24 or HLA-A2.
[0226] In another embodiment, the present invention also provides
the use of an active ingredient in the manufacture of a
pharmaceutical composition or agent for treating and/or preventing
cancer or tumor, and/or preventing a post-operative recurrence
thereof, said active ingredient selected from among:
(a) a peptide of the present invention; (b) a polynucleotide
encoding such a peptide of the present invention in an expressible
form; (c) an APC presenting a peptide of the present invention on
its surface; (d) an exosome presenting a peptide of the present
invention on its surface; and (e) a cytotoxic T cell of the present
invention.
[0227] Alternatively, the present invention further provides an
active ingredient for use in either or both of the treatment and
prevention of cancers or tumors, and/or prevention of a
post-operative recurrence thereof, said active ingredient selected
from among:
[0228] (a) a peptide of the present invention:
[0229] (b) a polynucleotide encoding such a peptide of the present
invention in an expressible form:
[0230] (c) an APC presenting a peptide of the present invention on
its surface:
[0231] (d) an exosome presenting a peptide of the present invention
on its surface; and
[0232] (e) a cytotoxic T cell of the present invention.
[0233] Alternatively, the present invention further provides a
method or process for the manufacture of a pharmaceutical
composition or agent for treating and/or preventing a cancer or
tumor, and/or preventing of a post-operative recurrence thereof,
wherein the method or process includes the step of formulating a
pharmaceutically or physiologically acceptable carrier with an
active ingredient selected from among:
[0234] (a) a peptide of the present invention;
[0235] (b) a polynucleotide encoding such a peptide of the present
invention in an expressible form;
[0236] (c) an APC presenting a peptide of the present invention on
its surface;
[0237] (d) an exosome presenting a peptide of the present invention
on its surface; and
[0238] (e) a cytotoxic T cell of the present invention.
[0239] In another embodiment, the present invention also provides a
method or process for the manufacture of a pharmaceutical
composition or agent for treating and/or preventing a cancer or
tumor, and/or preventing of a post-operative recurrence thereof,
wherein the method or process includes the steps of admixing an
active ingredient with a pharmaceutically or physiologically
acceptable carrier, wherein the active ingredient is selected from
among:
[0240] (a) a peptide of the present invention:
[0241] (b) a polynucleotide encoding such a peptide of the present
invention in an expressible form;
[0242] (c) an APC presenting a peptide of the present invention on
its surface;
[0243] (d) an exosome presenting a peptide of the present invention
on its surface; and
[0244] (e) a cytotoxic T cell of the present invention.
[0245] In another embodiment, the present invention also provides a
method for treating and/or preventing cancer or tumor, and/or
preventing a post-operative recurrence thereof, wherein the method
comprises the step of administering to a subject at least one
active ingredient selected from among:
[0246] (a) a peptide of the present invention;
[0247] (b) a polynucleotide encoding such a peptide of the present
invention in an expressible form;
[0248] (c) an APC presenting a peptide of the present invention on
its surface;
[0249] (d) an exosome presenting a peptide of the present invention
on its surface; and
[0250] (e) a cytotoxic T cell of the present invention.
[0251] According to the present invention, peptides having an amino
acid sequence selected from among SEQ ID NOs: 2 to 40 can be
HLA-A24 restricted epitope peptides. Among these peptides, peptides
having an amino acid sequence selected from among SEQ ID NOs: 2, 3,
6, 27 and 28 can effectively induce potent and specific immune
response against cancer expressing HLA-A24 and TOPK in a subject.
Likewise, the peptides having an amino acid sequence selected from
among SEQ ID NOs: 42 to 84 can be HLA-A2 restricted epitope
peptides. Among these peptides, peptides having an amino acid
sequence selected from among SEQ ID NOs: 42, 45, 47, 50, 51, 53,
54, 62, 63, 64, 66, 71, 72 and 76 can effectively induce potent and
specific immune response against cancer expressing HLA-A2 and TOPK
in a subject. Therefore, the pharmaceutical compositions or agents
which include any of peptides with the amino acid sequence selected
from among SEQ ID NOs: 2 to 40 (especially SEQ ID NOs: 2, 3, 6, 27
and 28) and modified peptides thereof are particularly suited for
the administration to subjects whose HLA antigen is HLA-A24.
Likewise, the pharmaceutical compositions or agents which include
any of peptides with the amino acid sequence selected from among
SEQ ID NOs: 42 to 84 (especially SEQ ID NOs: 42, 45, 47, 50, 51,
53, 54, 62, 63, 64, 66, 71, 72 and 76) modified peptides thereof
are particularly suited for the administration to subjects whose
HLA antigen is HLA-A2. The same applies to pharmaceutical
compositions or agents that contain polynucleotides encoding any of
these peptides (i.e., the polynucleotides of the present
invention).
[0252] Cancers to be treated by the pharmaceutical compositions or
agents of the present invention include all kinds of cancers
wherein TOPK is involved, including, but not limited to, AML,
bladder cancer, breast cancer, cervical cancer, cholangiocellular
carcinoma, colorectal cancer, diffuse-type gastric cancer. NSCLC,
lymphoma, osteosarcoma, prostate cancer, renal carcinoma, SCLC and
soft tissue tumor.
[0253] The pharmaceutical compositions or agents of the present
invention can contain in addition to the aforementioned active
ingredients, other peptides that have the ability to induce CTLs
against cancerous cells, other polynucleotides encoding the other
peptides, other cells that present the other peptides, and the
like. Examples of such "other" peptides having the ability to
induce CTLs against cancerous cells include, but are not limited
to, cancer specific antigens (e.g., identified TAAs).
[0254] If necessary, the pharmaceutical compositions or agents of
the present invention can optionally include other therapeutic
substances as an additional active ingredient, so long as the
substance does not inhibit the anti-tumoral effect of the active
ingredient. e.g., any of the peptides of the present invention. For
example, formulations can include anti-inflammatory substances,
pain killers, chemotherapeutics, and the like. In addition to
including other therapeutic substances in the medicament itself,
the medicaments of the present invention can also be administered
sequentially or concurrently with one or more other pharmacologic
compositions. The amounts of medicament and pharmacologic
composition depend, for example, on what type of pharmacologic
composition(s) is/are used, the disease being treated, and the
schedule and routes of administration.
[0255] Those of skill in the art will recognize that, in addition
to the ingredients particularly mentioned herein, the
pharmaceutical compositions or agents of the present invention can
include other substances conventional in the art having regard to
the type of formulation in question (e.g., fillers, binders,
diluents, excipients, etc.).
[0256] In one embodiment of the present invention, the
pharmaceutical compositions or agents of the present invention can
be included in articles of manufacture and kits containing
materials useful for treating the pathological conditions of the
disease to be treated, e.g., cancer. The article of manufacture can
include a container of any of the present pharmaceutical
compositions or agents with a label. Suitable containers include
bottles, vials, and test tubes. The containers can be formed from a
variety of materials, such as glass or plastic. The label on the
container should indicate the composition or agent is used for
treating or prevention of one or more conditions of the disease.
The label can also indicate directions for administration and so
on.
[0257] In addition to the container described above, a kit
including a pharmaceutical composition or agent of the present
invention can optionally further include a second container housing
a pharmaceutically-acceptable diluent. It can further include other
materials desirable from a commercial and user standpoint,
including other buffers, diluents, filters, needles, syringes, and
package inserts with instructions for use.
[0258] The pharmaceutical compositions or agents can, if desired,
be packaged in a pack or dispenser device which can contain one or
more unit dosage forms containing the active ingredient. The pack
can, for example, include metal or plastic foil, such as a blister
pack. The pack or dispenser device can be accompanied by
instructions for administration.
[0259] (1) Pharmaceutical Agents or Compositions Containing
Peptides as the Active Ingredient:
[0260] The peptides of this invention can be administered directly
as a pharmaceutical composition or agent, or if necessary, may be
formulated by conventional formulation methods. In the latter case,
in addition to the peptides of this invention, carriers,
excipients, and such that are ordinarily used for drugs can be
included as appropriate without particular limitations. Examples of
such carriers include, but are not limited to, sterilized water,
physiological saline, phosphate buffer, culture fluid and such.
Furthermore, the pharmaceutical compositions or agents can contain
as necessary, stabilizers, suspensions, preservatives, surfactants
and such. The pharmaceutical compositions or agents of the present
invention can be used for anticancer purposes.
[0261] The peptides of the present invention can be prepared as a
combination composed of two or more of peptides of the present
invention, to induce CTLs in vivo. The peptide combination can take
the form of a cocktail or can be conjugated to each other using
standard techniques. For example, the peptides can be chemically
linked or expressed as a single fusion polypeptide sequence. The
peptides in the combination can be the same or different. By
administering the peptides of the present invention, the peptides
are presented at a high density by the HLA antigens on APCs, then
CTLs that specifically react toward the complex formed between the
displayed peptide and the HLA antigen are induced. Alternatively,
APCs (e.g., DCs) are removed from subjects and then stimulated by
the peptides of the present invention to obtain APCs that present
any of the peptides of the present invention on their cell surface.
These APCs are readministered to the subjects to induce CTLs in the
subjects, and as a result, aggressiveness towards the
tumor-associated endothelium can be increased.
[0262] The pharmaceutical compositions or agents for the treatment
and/or prevention of cancer containing any peptide of the present
invention as the active ingredient can also include an adjuvant
known to effectively establish cellular immunity. Alternatively,
the pharmaceutical compositions or agents can be administered with
other active ingredients, or administered by formulation into
granules. An adjuvant refers to a compound that enhances the immune
response against the protein when administered together (or
successively) with the protein having immunological activity.
Adjuvants contemplated herein include those described in the
literature (Clin Microbiol Rev 1994, 7: 277-89). Examples of
suitable adjuvants include, but are not limited to, aluminum
phosphate, aluminum hydroxide, alum, cholera toxin, salmonella
toxin, IFA (Incomplete Freund's adjuvant), CFA (Complete Freund's
adjuvant), ISCOMatrix, GM-CSF, CpG, O/W emulsion and the like.
Furthermore, liposome formulations, granular formulations in which
the peptide is bound to few-micrometers diameter beads, and
formulations in which a lipid is bound to the peptide may be
conveniently used.
[0263] In another embodiment of the present invention, the peptides
of the present invention may also be administered in the form of a
pharmaceutically acceptable salt. Examples of preferred salts
include salts with an alkali metal, salts with a metal, salts with
an organic base, salts with an organic acid (e.g., acetic acid,
formic acid, propionic acid, fumaric acid, maleic acid, succinic
acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic
acid, methanesulfonic acid and so on) and salts with an inorganic
acid (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid,
sulfuric acid and so on). As used herein, the phrase
"pharmaceutically acceptable salt" refers to those salts that
retain the biological effectiveness and properties of the compound
and which are obtained by reaction with inorganic acids or bases
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid and the like.
[0264] In some embodiments, the pharmaceutical compositions or
agents of the present invention may further include a component
that primes CTLs. Lipids have been identified as substances capable
of priming CTLs in vive against viral antigens. For example,
palmitic acid residues can be attached to the epsilon- and
alpha-amino groups of a lysine residue and then linked to a peptide
of the present invention. The lipidated peptide can then be
administered either directly in a micelle or particle, incorporated
into a liposome, or emulsified in an adjuvant. As another example
of lipid priming of CTL responses, E. coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can be used
to prime CTLs when covalently attached to an appropriate peptide
(see, e.g., Deres et al., Nature 1989, 342: 561-4).
[0265] Examples of suitable methods of administration include, but
are not necessarily limited to, oral, intradermal, subcutaneous,
intramuscular, intraosseous, peritoneal, and intravenous injection,
or such, and systemic administration or local administration to the
vicinity of the targeted sites (i.e., direct injection). The
administration can be performed by single administration or boosted
by multiple administrations. The dose of the peptides of the
present invention can be adjusted appropriately according to the
disease to be treated, age of the patient, weight, method of
administration, and such, and is ordinarily 0.001 mg to 1000 mg,
for example, 0.01 mg to 100 mg, for example, 0.1 mg to 10 mg, for
example, 0.5 mg to 5 mg, and can be administered once in a few days
to few months. One skilled in the art can readily determine
suitable and optimal dosages.
[0266] (2) Pharmaceutical Agents or Compositions Containing
Polynucleotides as Active Ingredient:
[0267] The pharmaceutical compositions or agents of the present
invention can also contain nucleic acids encoding the peptides of
the present invention in an expressible form. Herein, the phrase
"in an expressible form" means that the polynucleotide, when
introduced into a cell, will be expressed in vivo as a polypeptide
that induces anti-tumor immunity. In an illustrative embodiment,
the nucleic acid sequence of the polynucleotide of interest
includes regulatory elements necessary for expression of the
polynucleotide. The polynucleotide(s) can be equipped so to achieve
stable insertion into the genome of the target cell (see. e.g.,
Thomas K R & Capecchi M R, Cell 1987, 51: 503-12 for a
description of homologous recombination cassette vectors). See,
e.g., Wolff et al., Science 1990, 247: 1465-8; U.S. Pat. Nos.
5,580,859, 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;
and WO 98/04720. Examples of DNA-based delivery technologies
include "naked DNA", facilitated (bupivacaine, polymers,
peptide-mediated) delivery, cationic lipid complexes, and
particle-mediated ("gene gun") or pressure-mediated delivery (see.
e.g., U.S. Pat. No. 5,922,687).
[0268] The peptides of the present invention can also be expressed
by viral or bacterial vectors. Examples of expression vectors
include attenuated viral hosts, such as vaccinia or fowlpox. This
approach involves the use of vaccinia virus. e.g., as a vector to
express nucleotide sequences that encode the peptide. Upon
introduction into a host, the recombinant vaccinia virus expresses
the immunogenic peptide, and thereby elicits an immune response.
Vaccinia vectors and methods useful in immunization protocols are
described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG
(Bacille Calmette Guerin). BCG vectors are described in Stover et
al., Nature 1991, 351: 456-60. A wide variety of other vectors
useful for therapeutic administration or immunization e.g., adeno
and adeno-associated virus vectors, retroviral vectors, Salmonella
typhi vectors, detoxified anthrax toxin vectors, and the like, will
be apparent. See, e.g., Shata et al, Mol Med Today 2000, 6: 66-71;
Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In
Vivo 2000, 14: 571-85.
[0269] Delivery of a polynucleotide into a patient can be either
direct, in which case the patient is directly exposed to a
polynucleotide-carrying vector, or indirect, in which case, cells
are first transformed with the polynucleotide of interest in vitro,
then the cells are transplanted into the patient. Theses two
approaches are known, respectively, as in vivo and ex vivo gene
therapies.
[0270] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu,
Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol
1993, 33: 573-96; Mulligan, Science 1993, 260: 926-32; Morgan &
Anderson, Ann Rev Biochem 1993, 62: 191-217; Trends in
Biotechnology 1993, 11(5): 155-215). Methods commonly known in the
art of recombinant DNA technology that are applicable to the
present invention are described by Ausubel et al. in Current
Protocols in Molecular Biology (John Wiley & Sons, N Y, 1993);
and Krieger in Gene Transfer and Expression, A Laboratory Manual
(Stockton Press, N Y, 1990).
[0271] Like administration of peptides, administration of
polynucleotides may be performed by oral, intradermal,
subcutaneous, intravenous, intramuscular, intraosseous, and/or
peritoneal injection, or such, and via systemic administration or
local administration to the vicinity of the targeted sites finds
use. The administration can be performed by single administration
or boosted by multiple administrations. The dose of the
polynucleotide in the suitable carrier or cells transformed with
the polynucleotide encoding the peptides of the present invention
can be adjusted appropriately according to the disease to be
treated, age of the patient, weight, method of administration, and
such, and is ordinarily 0.001 mg to 1000 mg, for example, 0.01 mg
to 100 mg, for example, 0.1 mg to 10 mg, for example, 0.5 mg to 5
mg, and can be administered once every a few days to once every few
months. One skilled in the art can readily determine suitable and
optimal dosages.
X. METHODS OF USING THE PEPTIDES, POLYNUCLEOTIDE, EXOSOMES, APCS
AND CTLS
[0272] The peptides and polynucleotides of the present invention
can be used for preparing or inducing APCs and CTLs. The exosomes
and APCs of the present invention can be also used for preparing or
inducing CTLs. The peptides, polynucleotides, exosomes and APCs can
be used in combination with any other compounds so long as the
additional compounds do not inhibit CTL inducibility. Thus, any of
the aforementioned pharmaceutical compositions or agents of the
present invention can be used for preparing or inducing CTLs. In
addition thereto, those including the peptides or polynucleotides
can be also used for preparing or inducing APCs as discussed
below.
[0273] (1) Method of Inducing Antigen-Presenting Cells (APCs)
[0274] The present invention provides methods of inducing APCs with
high CTL inducibility using the peptides or polynucleotides of the
present invention.
[0275] The methods of the present invention include the step of
contacting APCs with the peptides of the present invention in
vitro, ex vivo or in vivo. For example, the method of inducing APCs
ex vivo can include steps of:
[0276] a: collecting APCs from a subject, and
[0277] b: contacting the APCs of step a with the peptide of the
present invention.
[0278] The APCs are not limited to a particular kind of cells.
Examples of APCs include, but are not limited to, DCs, Langerhans
cells, macrophages, B cells, and activated T cells, which are known
to present proteinaceous antigens on their cell surface so as to be
recognized by lymphocytes. Preferably, DCs can be used since they
have the strongest CTL inducibility among APCs. Any one of peptides
of the present invention can be used by itself or in combination
with other peptides of the present invention or CTL-inducible
peptides derived from TAAs other than TOPK.
[0279] On the other hand, when the peptides of the present
invention are administered to a subject, the APCs are contacted
with the peptides in vivo, and consequently, the APCs with CTL
inducibility are induced in the body of the subject. Thus, the
method of the present invention may include the step of
administering a peptide of the present invention to a subject to
induce an APC with CTL inducibility in the body of the subject.
Similarly, when the polynucleotides of this invention are
administered to a subject in an expressible form, the peptides of
the present invention are expressed and contacted with APCs in
vivo, and consequently, the APCs with CTL inducibility are induced
in the body of the subject. Thus, the methods of the present
invention may include the step of administering a polynucleotide of
the present invention to a subject to induce an APC with CTL
inducibility in the body of the subject. The phrase "expressible
form" was described above in section "IX. Pharmaceutical Agents or
Compositions (2) Pharmaceutical Agents or Compositions Containing
Polynucleotides as the Active Ingredient".
[0280] Alternatively, the methods of the present invention may
include the step of introducing a polynucleotide encoding the
peptide of the present invention into an APC to induce an APC with
CTL inducibility. For example, the method can include steps of:
[0281] a: collecting APCs from a subject, and
[0282] b: introducing a polynucleotide encoding the peptide of the
present invention into the APC of step a.
[0283] Step b can be performed as described above in section "VI.
Antigen-Presenting Cells".
[0284] Alternatively, the methods of the present invention may
include the step of preparing an antigen-presenting cell (APC) that
can specifically induce CTL activity against TOPK, via one of the
following steps:
[0285] (a) contacting an APC with a peptide of the present
invention in vitro, ex vivo or in vivo; and
[0286] (b) introducing a polynucleotide encoding a peptide of the
present invention into an APC.
[0287] Alternatively, the methods of the present invention may
serve to induce an APC having CTL inducibility, such methods
including a step selected from among:
[0288] (a) contacting an APC with the peptide of the present
invention:
[0289] (b) introducing the polynucleotide encoding the peptide of
the present invention into an APC.
[0290] In a preferred embodiment, the present invention provides
the method of inducing or preparing an APC having CTL inducibility,
such method including one of the following steps:
(a) contacting an APC expressing HLA-A24 with a peptide having an
amino acid sequence selected from among SEQ ID NOs: 2 to 40
(especially SEQ ID NOs: 2, 3, 6, 27 and 28) or modified peptide
thereof in vitro, ex vivo or in vivo; and (b) introducing a
polynucleotide encoding a peptide having an amino acid sequence
selected from among SEQ ID NOs: 2 to 40 (especially SEQ ID NOs: 2,
3, 6, 27 and 28) or modified peptide thereof into an APC expressing
HLA-A2. APCs induced by the above method present such peptides via
HLA-A24 on their surface, and can induce CTLs having specific
cytotoxic activity against cells expressing HLA-A24 and TOPK.
[0291] In another embodiment, the present invention provides the
method of inducing or preparing an APC having CTL inducibility,
such method including one of the following steps:
[0292] (a) contacting an APC expressing HLA-A2 with a peptide
having an amino acid sequence selected from among SEQ ID NOs: 42 to
84 (especially SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62, 63, 64,
66, 71, 72 and 76) or modified peptide thereof in vitro, ex vivo or
in vivo; and
[0293] (b) introducing a polynucleotide encoding a peptide having
an amino acid sequence selected from among SEQ ID NOs: SEQ ID NOs:
42 to 84 (especially SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62,
63, 64, 66, 71, 72 and 76) or modified peptide thereof into an APC
expressing HLA-A2.
[0294] APCs induced by the above method present such peptides via
HLA-A2 on their surface, and can induce CTLs having specific
cytotoxic activity against cells expressing HLA-A2 and TOPK.
[0295] The methods of the present invention can be carried out in
vitro, ex vive or in vivo. Preferably, the methods of the present
invention can be carried out in vitro or ex vivo. APCs used for
induction of APCs having CTL inducibility can be preferably APCs
expressing HLA-A24 or HLA-A2 antigen. Such APCs can be prepared by
the methods well-known in the arts from peripheral blood
mononuclear cells (PBMCs) obtained from a subject whose HLA antigen
is HLA-A24 or HLA-A2. The APCs induced by the method of the present
invention can be APCs that present a complex of the peptide of the
present invention and HLA antigen (HLA A24 or HLA-A2 antigen) in
its surface. When APCs induced by the method of the present
invention are administered to a subject in order to induce immune
responses against cancer in the subject, the subject is preferably
the same one from whom APCs are derived. However, the subject may
be a different one from the APC donor so long as the subject has
the same HLA type with the APC donor.
[0296] In another embodiment, the present invention provide agents
or compositions for use in inducing an APC having CTL inducibility,
and such agents or compositions include one or more peptides or
polynucleotides of the present invention.
In another embodiment, the present invention provides the use of
the peptide of the present invention or the polynucleotide encoding
the peptide in the manufacture of an agent or composition
formulated for inducing APCs. Alternatively, the present invention
further provides the peptide of the present invention or the
polypeptide encoding the peptide for use in inducing an APC having
CTL inducibility.
[0297] (2) Method of Inducing CTLs
[0298] The present invention also provides methods for inducing
CTLs using the peptides, polynucleotides, exosomes or APCs of the
present invention.
[0299] The present invention also provides methods for inducing
CTLs using a polynucleotide/polynucleotides encoding polypeptides
(i.e., TCR subunits) that are capable of forming a T cell receptor
(TCR) that is capable of recognizing a complex of the peptide of
the present invention and an HLA antigen. Preferably, the methods
for inducing CTLs include at least one step selected from
among:
[0300] a: contacting a CD8-positive T cell with an
antigen-presenting cell that presents on its surface a complex of
an HLA antigen and a peptide of the preset invention;
[0301] b: contacting a CD8-positive T cell with an exosome that
presents on its surface a complex of an HLA antigen and a peptide
of the present invention; and
[0302] c: introducing a polynucleotide/polynucleotides encoding
polypeptides that are capable of forming a TCR that is capable of
recognizing a complex of a peptide of the present invention and an
HLA antigen into a CDR-positive T cell.
[0303] When the peptides, polynucleotides. APCs, or exosomes of the
present invention are administered to a subject, CTLs are induced
in the body of the subject, and the strength of the immune response
targeting the cancer cells expressing TOPK is enhanced. Thus,
instead of the step aforementioned step, the methods of the present
invention may include the step of administering the peptides,
polynucleotides. APCs or exosomes of the present invention to a
subject.
[0304] Alternatively, CTLs can be also induced by using them ex
vivo or in vivo, and after inducing CTLs, the activated CTLs are
returned to the subject. For example, the method can include steps
of:
[0305] a: collecting APCs from subject,
[0306] b: contacting the APCs of step a, with the peptide of the
present invention, and
[0307] c: co-culturing the APCs of step b with CD8-positive T
cells.
[0308] The APCs to be co-cultured with the CD8-positive T cells in
above step c can also be prepared by transferring a polynucleotide
of the present invention into APCs as described above in section
"VI. Antigen-Presenting Cells", although the present invention is
not limited thereto and thus encompasses any APCs that effectively
present on its surface a complex of an HLA antigen and a peptide of
the present invention.
[0309] One may optionally utilize an exosome that presents on its
surface a complex of an HLA antigen and the peptide of the present
invention instead of the afore-mentioned APCs. Namely, the present
invention can includes the step of co-culturing exosomes presenting
on its surface a complex of an HLA antigen and the peptide of the
present invention. Such exosomes can be prepared by the methods
described above in section "V. Exosomes". Suitable APCs and
exosomes for the method of the present invention present a complex
of the peptide of the present invention and HLA-A24 or HLA-A2 on
its surface. For example, an APC or exosome that present a complex
of an HLA-A24 and a peptide having an amino acid sequence selected
from among SEQ ID NOs: 2, 3, 6, 27 and 28 (or modified peptide
thereof) on its surface can be preferably utilize for inducing a
CTL having specific cytotoxic activity against a cell expressing
HLA-A24 and TOPK. Likewise, an APC or exosome that present a
complex of an HLA-A2 and a peptide having an amino acid sequence
selected from among SEQ ID NOs: 42, 45, 47, 50, 51, 53, 54, 62, 63,
64, 66, 71, 72 and 76 (or modified peptide thereof) on its surface
can be preferably utilize for inducing a CTL having specific
cytotoxic activity against a cell expressing HLA-A2 and TOPK.
[0310] Furthermore, the CTL of the present invention can be induced
by introducing into a CD8 positive T cell a
polynucleotide/polynucleotides encoding the TCR subunits, wherein
the TCR formed by such TCR subunits is capable of binding to a
complex of an HLA antigen and the peptide of the invention on a
cell surface. Such transduction can be performed as described above
in section "VIII. T Cell Receptor (TCR)".
[0311] The methods of the present invention can be carried out in
vitro, ex vivo or in vivo. Preferably, the methods of the present
invention can be carried out in vitro or ex vivo. CD8- positive T
cells used for induction of CTLs can be prepared by well-known
methods in the art from PBMCs obtained from a subject. In preferred
embodiments, the donor for CD8-positive T cells can be a subject
whose HLA antigen is HLA-A24 or HLA-A2. The CTLs induced by the
methods of the present invention can be CTLs that can recognize
cells presenting a complex of the peptide of the present invention
and HLA antigen on its surface. Such CTLs can show specific
cytotoxic activity against cells that present the peptide of the
present invention on its surface, and therefore, can show specific
cytotoxic activity against cells expressing TOPK (e.g., cancer
cells). When CTLs induced by the method of the present invention
are administered to a subject in order to induce immune responses
against cancer in the subject, the subject is preferably the same
one from whom CD8-positive T cells are derived. However, the
subject may be a different one from the CD8-positive T cell donor
so long as the subject has the same HLA type with the CD8-positive
T cell donor.
[0312] In addition, the present invention provides a method or
process for manufacturing a pharmaceutical composition or agent
that induces CTLs, wherein the method includes the step of admixing
or formulating the peptide of the present invention with a
pharmaceutically acceptable carrier.
[0313] In another embodiment, the present invention provide an
agent or composition for inducing a CTL, wherein the agent or
composition comprises one or more peptide(s), one or more
polynucleotide(s), or one or more APCs or exosomes of the present
invention.
[0314] In another embodiment, the present invention provides the
use of the peptide, the polynucleotide, or APC or exosome of the
present invention in the manufacture of an agent or composition
formulated for inducing a CTL
[0315] Alternatively, the present invention further provides the
peptide, the polynucleotide, or APC or exosome of the present
invention for use in inducing a CTL.
[0316] (3) Methods of Inducing Immune Response
[0317] Moreover, the present invention provides methods of inducing
immune response against diseases related to TOPK. Diseases
contemplated include cancer, examples of which include, but are not
limited to, AML, bladder cancer, breast cancer, cervical cancer,
cholangiocellular carcinoma, colorectal cancer, diffuse-type
gastric cancer, NSCLC, lymphoma, osteosarcoma, prostate cancer,
renal carcinoma, SCLC and soft tissue tumor.
[0318] The methods of the present invention may include the step of
administering agent(s) or composition(s) containing any of the
peptides of the present invention or polynucleotides encoding them.
Alternatively, the method of the present invention also includes
the step of administering exosomes or APCs presenting any of the
peptides of the present invention. For details, see the item of
"IX. Pharmaceutical Agents or Compositions", particularly the part
describing the use of the pharmaceutical compositions of the
present invention as vaccines. In addition, the exosomes and APCs
that can be employed for the present methods for inducing immune
response are described in detail under the items of "V. Exosomes",
"VI. Antigen-Presenting Cells (APCs)", and (1) and (2) of "X.
Methods of Using the Peptides, Exosomes, APCs and CTLs", supra.
[0319] The present invention also provides a method or process for
manufacturing a pharmaceutical composition or agent that induce an
immune response against cancer, wherein the method may include the
step of admixing or formulating a peptide or polynucleotide of the
present invention with a pharmaceutically acceptable carrier.
[0320] Alternatively, the method of the present invention may
include the step of administrating a vaccine or a pharmaceutical
composition or agent of the present invention that contains:
(a) a peptide of the present invention; (b) a polynucleotide
encoding the peptide of the present invention in an expressible
form; (c) an APC presenting a peptide of the present invention on
its surface; (d) an exosome presenting a peptide of the present
invention on its surface; or (d) a cytotoxic T cell of the present
invention.
[0321] In the context of the present invention, a cancer
over-expressing TOPK can be treated with these active ingredients.
Examples of such cancer include, but are not limited to, AML,
bladder cancer, breast cancer, cervical cancer, cholangiocellular
carcinoma, colorectal cancer, diffuse-type gastric cancer, NSCLC,
lymphoma, osteosarcoma, prostate cancer, renal carcinoma, SCLC and
soft tissue tumor. Accordingly, prior to the administration of the
vaccines or pharmaceutical compositions or agents including the
aforementioned active ingredients, it is preferable to confirm
whether the expression level of TOPK in the subject to be treated
is enhanced. Thus, in one embodiment, the present invention
provides a method for treating cancer (over)expressing TOPK in a
patient in need thereof, such method including the steps of:
[0322] i) determining the expression level of TOPK in biological
sample(s) obtained from a subject with the cancer to be
treated;
[0323] ii) comparing the expression level of TOPK with normal
control; and
[0324] iii) administrating at least one component selected from
among (a) to (d) described above to a subject with cancer
over-expressing TOPK as compared with normal control.
[0325] Alternatively, the present invention provides a vaccine or
pharmaceutical composition including at least one component
selected from among (a) to (d) described above, to be administered
to a subject having cancer over-expressing TOPK. In other words,
the present invention further provides a method for identifying a
subject to be treated with the TOPK polypeptide of the present
invention, such method including the step of determining an
expression level of TOPK in subject-derived biological sample(s),
wherein an increase of the level compared to a normal control level
of the gene indicates that the subject may have cancer which may be
treated with the TOPK polypeptide of the present invention. The
methods of treating cancer of the present invention will be
described in more detail in below.
[0326] Any subject-derived cell or tissue can be used for the
determination of TOPK expression so long as it includes the
objective transcription or translation product of TOPK. Examples of
suitable samples include, but are not limited to, bodily tissues
and fluids, such as blood, sputum and urine. Preferably, the
subject-derived cell or tissue sample contains a cell population
including an epithelial cell, more preferably a cancerous
epithelial cell or an epithelial cell derived from tissue suspected
to be cancerous. Further, if necessary, the cell may be purified
from the obtained bodily tissues and fluids, and then used as the
subjected-derived sample.
A subject to be treated by the present method is preferably a
mammal. Illustrative mammals include, but are not limited to, e.g.,
human, non-human primate, mouse, rat, dog, cat, horse, and cow.
[0327] According to the present invention, the expression level of
TOPK in biological sample obtained from a subject may be
determined. The expression level can be determined at the
transcription (nucleic acid) product level, using methods known in
the art. For example, the mRNA of TOPK may be quantified using
probes by hybridization methods (e.g., Northern hybridization). The
detection may be carried out on a chip or an array. The use of an
array is preferable for detecting the expression level of TOPK.
Those skilled in the art can prepare such probes utilizing the
sequence information of TOPK. For example, the cDNA of TOPK may be
used as the probes. If necessary, the probes may be labeled with a
suitable label, such as dyes, fluorescent substances and isotopes,
and the expression level of the gene may be detected as the
intensity of the hybridized labels.
[0328] Furthermore, the transcription product of TOPK may be
quantified using primers by amplification-based detection methods
(e.g., RT-PCR). Such primers may be prepared based on the available
sequence information of the gene.
[0329] Specifically, a probe or primer used for the present method
hybridizes under stringent, moderately stringent, or low stringent
conditions to the mRNA of TOPK. As used herein, the phrase
"stringent (hybridization) conditions" refers to conditions under
which a probe or primer will hybridize to its target sequence, but
not to other sequences. Stringent conditions are sequence-dependent
and will be different under different circumstances. Specific
hybridization of longer sequences is observed at higher
temperatures than shorter sequences. Generally, the temperature of
a stringent condition is selected to be about 5 degree Centigrade
lower than the thermal melting point (Tm) for a specific sequence
at a defined ionic strength and pH. The Tm is the temperature
(under a defined ionic strength, pH and nucleic acid concentration)
at which 50% of the probes complementary to their target sequence
hybridize to the target sequence at equilibrium. Since the target
sequences are generally present at excess, at Tm, 50% of the probes
are occupied at equilibrium. Typically, stringent conditions will
be those in which the salt concentration is less than about 1.0 M
sodium ion, typically about 0.01 to 1.0 M sodium ion (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about 30
degree Centigrade for short probes or primers (e.g., 10 to 50
nucleotides) and at least about 60 degree Centigrade for longer
probes or primers. Stringent conditions may also be achieved with
the addition of destabilizing substances, such as formamide.
[0330] A probe or primer of the present invention is typically a
substantially purified oligonucleotide. The oligonucleotide
typically includes a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 2000, 1000, 500, 400,
350, 300, 250, 200, 150, 100, 50, or 25, consecutive sense strand
nucleotide sequence of a nucleic acid including a TOPK sequence, or
an anti sense strand nucleotide sequence of a nucleic acid
including a TOPK sequence, or of a naturally occurring mutant of
these sequences. In particular, for example, in a preferred
embodiment, an oligonucleotide having 5-50 in length can be used as
a primer for amplifying the genes, to be detected. More preferably,
mRNA or cDNA of a TOPK gene can be detected with oligonucleotide
probe or primer of a specific size, generally 15-30b in length. The
size may range from at least 10 nucleotides, at least 12
nucleotides, at least 15 nucleotides, at least 20 nucleotides, at
least 25 nucleotides, at least 30 nucleotides and the probes and
primers may range in size from 5-10 nucleotides, 10-15 nucleotides,
15-20 nucleotides, 20-25 nucleotides and 25-30 nucleotides. In
preferred embodiments, length of the oligonucleotide probe or
primer can be selected from 15-25. Assay procedures, devices, or
reagents for the detection of gene by using such oligonucleotide
probe or primer are well known (e.g. oligonucleotide microarray or
PCR). In these assays, probes or primers can also include tag or
linker sequences. Further, probes or primers can be modified with
detectable label or affinity ligand to be captured. Alternatively,
in hybridization based detection procedures, a polynucleotide
having a few hundreds (e.g., about 100-200) bases to a few kilo
(e.g., about 1000-2000) bases in length can also be used for a
probe (e.g., northern blotting assay or cDNA microarray
analysis).
[0331] Alternatively, the translation product may be detected for
the diagnosis of the present invention. For example, the quantity
of TOPK protein (SEQ ID NO: 86) or the immunologically fragment
thereof may be determined. Methods for determining the quantity of
the protein as the translation product include immunoassay methods
that use an antibody specifically recognizing the protein. The
antibody may be monoclonal or polyclonal. Furthermore, any fragment
or modification (e.g., chimeric antibody, scFv, Fab, F(ab').sub.2,
Fv, etc.) of the antibody may be used for the detection, so long as
the fragment or modified antibody retains the binding ability to
the TOPK protein. Such antibodies against the peptides of the
present invention and the fragments thereof are also provided by
the present invention. Methods to prepare these kinds of antibodies
for the detection of proteins are well known in the art, and any
method may be employed in the present invention to prepare such
antibodies and equivalents thereof.
[0332] As another method to detect the expression level of TOPK
gene based on its translation product, the intensity of staining
may be measured via immunohistochemical analysis using an antibody
against the TOPK protein. Namely, in this measurement, strong
staining indicates increased presence/level of the protein and, at
the same time, high expression level of TOPK gene.
[0333] The expression level of a target gene, e.g., the TOPK gene,
in cancer cells can be determined to be increased if the level
increases from the control level (e.g., the level in normal cells)
of the target gene by, for example, 10%, 25%, or 50%; or increases
to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more
than 5.0 fold, more than 10.0 fold, or more.
[0334] The control level may be determined at the same time as the
cancer cells by using a sample(s) previously collected and stored
from a subject/subjects whose disease state(s) (cancerous or
non-cancerous) is/are known. In addition, normal cells obtained
from non-cancerous regions of an organ that has the cancer to be
treated may be used as normal control. Alternatively, the control
level may be determined by a statistical method based on the
results obtained by analyzing previously determined expression
level(s) of TOPK gene in samples from subjects whose disease states
are known. Furthermore, the control level can be derived from a
database of expression patterns from previously tested cells.
Moreover, according to an aspect of the present invention, the
expression level of TOPK gene in a biological sample may be
compared to multiple control levels, which are determined from
multiple reference samples. It is preferred to use a control level
determined from a reference sample derived from a tissue type
similar to that of the subject-derived biological sample. Moreover,
it is preferred to use the standard value of the expression levels
of TOPK gene in a population with a known disease state. The
standard value may be obtained by any method known in the art. For
example, a range of mean+/-2 S.D. or mean+/-3 S.D. may be used as
the standard value.
[0335] In the context of the present invention, a control level
determined from a biological sample that is known to be
non-cancerous is referred to as a "normal control level". On the
other hand, if the control level is determined from a cancerous
biological sample, it is referred to as a "cancerous control
level". Difference between a sample expression level and a control
level can be normalized to the expression level of control nucleic
acids, e.g., housekeeping genes, whose expression levels are known
not to differ depending on the cancerous or non-cancerous state of
the cell. Exemplary control genes include, but are not limited to,
beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal
protein P1.
[0336] When the expression level of TOPK gene is increased as
compared to the normal control level, or is similar/equivalent to
the cancerous control level, the subject may be diagnosed with
cancer to be treated.
[0337] The present invention also provides a method of (i)
diagnosing whether a subject suspected to have cancer to be
treated, and/or (ii) selecting a subject for cancer treatment, such
method including the steps of:
a) determining the expression level of TOPK in biological sample(s)
obtained from a subject who is suspected to have the cancer to be
treated: b) comparing the expression level of TOPK with a normal
control level; c) diagnosing the subject as having the cancer to be
treated, if the expression level of TOPK is increased as compared
to the normal control level; and d) selecting the subject for
cancer treatment, if the subject is diagnosed as having the cancer
to be treated, in step c).
[0338] Alternatively, such a method may include the steps of:
[0339] a) determining the expression level of TOPK in biological
sample(s) obtained from a subject who is suspected to have the
cancer to be treated;
[0340] b) comparing the expression level of TOPK with a cancerous
control level:
[0341] c) diagnosing the subject as having the cancer to be
treated, if the expression level of TOPK is similar or equivalent
to the cancerous control level; and
[0342] d) selecting the subject for cancer treatment, if the
subject is diagnosed as having the cancer to be treated, in step
c).
[0343] The present invention also provides a diagnostic kit for
diagnosing or determining a subject who is or is suspected to be
suffering from or at risk of developing a cancer that can be
treated with the TOPK polypeptide of the present invention, which
may also be useful in either or both of assessing and monitoring
the efficacy, or applicability of a cancer immunotherapy.
Preferably, the cancer includes, but is not limited to, AML,
bladder cancer, breast cancer, cervical cancer, cholangiocellular
carcinoma, colorectal cancer, diffuse-type gastric cancer, NSCLC,
lymphoma, osteosarcoma, prostate cancer, renal carcinoma, SCLC and
soft tissue tumor. More particularly, the kit preferably includes
at least one reagent for detecting the expression of the TOPK gene
in a subject-derived cell, which reagent may be selected from the
group of:
[0344] (a) a reagent for detecting an mRNA of the TOPK gene:
[0345] (b) a reagent for detecting the TOPK protein or the
immunologically fragment thereof; and
[0346] (c) a reagent for detecting the biological activity of the
TOPK protein.
[0347] Examples of reagents suitable for detecting an mRNA of the
TOPK gene include nucleic acids that specifically bind to or
identify the TOPK mRNA, such as oligonucleotides that have a
complementary sequence to a portion of the TOPK mRNA. These kinds
of oligonucleotides are exemplified by primers and probes that are
specific to the TOPK mRNA. These kinds of oligonucleotides may be
prepared based on methods well known in the art. If needed, the
reagent for detecting the TOPK mRNA may be immobilized on a solid
matrix. Moreover, more than one reagent for detecting the TOPK mRNA
may be included in the kit.
[0348] On the other hand, examples reagents suitable for detecting
the TOPK protein or the immunologically fragment thereof may
include antibodies to the TOPK protein or the immunologically
fragment thereof. The antibody may be monoclonal or polyclonal.
Furthermore, any fragment or modification (e.g., chimeric antibody,
scFv, Fab, F(ab').sub.2, Fv, etc.) of the antibody may be used as
the reagent, so long as the fragment or modified antibody retains
the binding ability to the TOPK protein or the immunologically
fragment thereof. Methods to prepare these kinds of antibodies for
the detection of proteins are well known in the art, and any method
may be employed in the present invention to prepare such antibodies
and equivalents thereof. Furthermore, the antibody may be labeled
with signal generating molecules via direct linkage or an indirect
labeling technique. Labels and methods for labeling antibodies and
detecting the binding of the antibodies to their targets are well
known in the art, and any labels and methods may be employed for
the present invention. Moreover, more than one reagent for
detecting the TOPK protein may be included in the kit.
[0349] The kit may contain more than one of the aforementioned
reagents. The kit can further include a solid matrix and reagent
for binding a probe against a TOPK gene or antibody against a TOPK
peptide, a medium and container for culturing cells, positive and
negative control reagents, and a secondary antibody for detecting
an antibody against a TOPK peptide. For example, tissue samples
obtained from subjects without cancer or suffering from cancer, may
serve as useful control reagents. A kit of the present invention
may further include other materials desirable from a commercial and
user standpoint, including buffers, diluents, filters, needles,
syringes, and package inserts (e.g., written, tape. CD-ROM, etc.)
with instructions for use. These reagents and such may be retained
in a container with a label. Suitable containers include bottles,
vials, and test tubes. The containers may be formed from a variety
of materials, such as glass or plastic.
[0350] In an embodiment of the present invention, when the reagent
is a probe against the TOPK mRNA, the reagent may be immobilized on
a solid matrix, such as a porous strip, to form at least one
detection site. The measurement or detection region of the porous
strip may include a plurality of sites, each containing a nucleic
acid (probe). A test strip may also contain sites for negative
and/or positive controls. Alternatively, control sites may be
located on a strip separated from the test strip. Optionally, the
different detection sites may contain different amounts of
immobilized nucleic acids, i.e., a higher amount in the first
detection site and lesser amounts in subsequent sites. Upon the
addition of a test sample, the number of sites displaying a
detectable signal provides a quantitative indication of the amount
of TOPK mRNA present in the sample. The detection sites may be
configured in any suitably detectable shape and are typically in
the shape of a bar or dot spanning the width of a test strip.
[0351] The kit of the present invention may further include a
positive control sample or TOPK standard sample. The positive
control sample of the present invention may be prepared by
collecting TOPK positive samples and then assaying their TOPK
levels. Alternatively, a purified TOPK protein or polynucleotide
may be added to cells that do not express TOPK to form the positive
sample or the TOPK standard sample. In the present invention,
purified TOPK may be a recombinant protein. The TOPK level of the
positive control sample is, for example, more than the cut off
value.
[0352] In one embodiment, the present invention further provides a
diagnostic kit including, a protein or a partial protein thereof
specifically recognized by the antibody of the present invention or
the fragment thereof.
[0353] Examples of the partial peptide of the protein of the
present invention include polypeptides composed of at least 8,
preferably 15, and more preferably 20 contiguous amino acids in the
amino acid sequence of the protein of the present invention. Cancer
can be diagnosed by detecting an antibody in a sample (e.g., blood,
tissue) using a protein or a peptide (polypeptide) of the present
invention. The method for preparing the protein of the present
invention and peptides are as described above.
[0354] The methods for diagnosing cancer of the present invention
can be performed by determining the difference between the amount
of anti-TOPK antibody and that in the corresponding control sample
as describe above. The subject is suspected to be suffering from
cancer, if cells or tissues of the subject contain antibodies
against the expression products (TOPK) of the gene and the quantity
of the anti-TOPK antibody is determined to be more than the cut off
value in level compared to that in normal control.
[0355] In another embodiment, a diagnostic kit of the present
invention may include the peptide of the present invention and an
HLA molecule binding thereto. The method for detecting antigen
specific CTLs using antigenic peptides and HLA molecules has
already been established (for example, Altman J D et al., Science.
1996, 274(5284): 94-6). Thus, the complex of the peptide of the
present invention and the HLA molecule can be applied to the
detection method to detect tumor antigen specific CTLs, thereby
enabling earlier detection, recurrence and/or metastasis of cancer.
Further, it can be employed for the selection of subjects
applicable with the pharmaceuticals including the peptide of the
present invention as an active ingredient, or the assessment of the
treatment effect of the pharmaceuticals.
[0356] Particularly, according to the known method (see, for
example, Altman J D et al., Science. 1996, 274(5284): 94-6), the
oligomer complex, such as tetramer, of the radiolabeled HLA
molecule and the peptide of the present invention can be prepared.
With using the complex, the diagnosis can be done, for example, by
quantifying the antigen-peptide specific CTLs in the peripheral
blood lymphocytes derived from the subject suspected to be
suffering from cancer.
[0357] The present invention further provides method and diagnostic
agents for evaluating immunological response of subject by using
peptide epitopes as described herein. In one embodiment of the
invention, HLA-A24 or HLA-A2 restricted peptides as described
herein are used as reagents for evaluating or predicting an immune
response of a subject. The immune response to be evaluated is
induced by contacting an immunogen with immunocompetent cells in
vitro or in vivo. In preferred embodiments, the immunocompetent
cells for evaluating an immunological response, may be selected
from among peripheral blood, peripheral blood lymphocyte (PBL), and
peripheral blood mononuclear cell (PBMC). Methods for collecting or
isolating such immunocompetent cells are well known in the arts. In
some embodiments, any agent that may result in the production of
antigen specific CTLs that recognize and bind to the peptide
epitope (s) may be employed as the reagent. The peptide reagent
need not be used as the immunogen. Assay systems that are used for
such an analysis include relatively recent technical developments
such as tetramers, staining for intracellular lymphokines and
interferon release assays, or ELISPOT assays. In a preferred
embodiment, immunocompetent cells to be contacted with peptide
reagent may be antigen presenting cells including dendritic
cells.
[0358] For example, peptides of the present invention may be used
in tetramer staining assays to assess peripheral blood mononuclear
cells for the presence of antigen-specific CTLs following exposure
to a tumor cell antigen or an immunogen. The HLA tetrameric complex
may be used to directly visualize antigen specific CTLs (see, e.
g., Ogg et al., Science 279: 2103-2106, 1998; and Altman et al,
Science 174: 94-96, 1996) and determine the frequency of the
antigen-specific CTL population in a sample of peripheral blood
mononuclear cells. A tetramer reagent using a peptide of the
invention may be generated as described below.
[0359] A peptide that binds to an HLA molecule is refolded in the
presence of the corresponding HLA heavy chain and beta
2-microglobulin to generate a trimolecular complex. In the complex,
carboxyl terminal of the heavy chain is biotinylated at a site that
was previously engineered into the protein. Then, streptavidin is
added to the complex to form tetramer composed of the trimolecular
complex and streptavidin. By means of fluorescently labeled
streptavidin, the tetramer can be used to stain antigen-specific
cells. The cells can then be identified, for example, by flow
cytometry. Such an analysis may be used for diagnostic or
prognostic purposes. Cells identified by the procedure can also be
used for therapeutic purposes.
[0360] The present invention also provides reagents to evaluate
immune recall responses (see, e. g., Bertoni et aL, J. Clin.
Invest. 100: 503-513, 1997 and Penna et aL. J Exp. Med. 174:
1565-1570, 1991) including peptides of the present invention. For
example, patient PBMC samples obtained from individuals with a
cancer to be treated are analyzed for the presence of
antigen-specific CTLs using specific peptides. A blood sample
containing mononuclear cells can be evaluated by cultivating the
PBMCs and stimulating the cells with a peptide of the invention.
After an appropriate cultivation period, the expanded cell
population can be analyzed, for example, for CTL activity.
[0361] The peptides may be also used as reagents to evaluate the
efficacy of a vaccine. PBMCs obtained from a patient vaccinated
with an immunogen may be analyzed using, for example, either of the
methods described above. The patient is HLA typed, and peptide
epitope reagents that recognize the allele specific molecules
present in that patient are selected for the analysis. The
immunogenicity of the vaccine may be indicated by the presence of
epitope-specific CTLs in the PBMC sample.
[0362] The peptides of the invention may be also used to make
antibodies, using techniques well known in the art (see, e. g.
CURRENT PROTOCOLS IMMUNOLOGY. Wiley/Greene, NY; and Antibodies A
Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory
Press, 1989), which may be useful as reagents to diagnose or
monitor cancer. Such antibodies may include those that recognize a
peptide in the context of an HLA molecule, i. e., antibodies that
bind to a peptide-MHC complex.
[0363] The peptides and compositions of the present invention have
a number of additional uses, some of which are described herein.
For instance, the present invention provides a method for
diagnosing or detecting a disorder characterized by expression of a
TOPK immunogenic polypeptide. These methods involve determining
expression of a TOPK HLA binding peptide, or a complex of a TOPK
HLA binding peptide and an HLA class I molecule in a biological
sample. The expression of a peptide or complex of peptide and HLA
class I molecule can be determined or detected by assaying with a
binding partner for the peptide or complex. In a preferred
embodiment, a binding partner for the peptide or complex is an
antibody recognizes and specifically bind to the peptide. The
expression of TOPK in a biological sample, such as a tumor biopsy,
can also be tested by standard PCR amplification protocols using
TOPK primers. An example of tumor expression is presented herein
and further disclosure of exemplary conditions and primers for TOPK
amplification can be found in WO2003/27322.
[0364] Preferably, the diagnostic methods involve contacting a
biological sample isolated from a subject with an agent specific
for the TOPK HLA binding peptide to detect the presence of the TOPK
HLA binding peptide in the biological sample. As used herein,
"contacting" means placing the biological sample in sufficient
proximity to the agent and under the appropriate conditions of, e.
g., concentration, temperature, time, ionic strength, to allow the
specific interaction between the agent and TOPK HLA binding peptide
that are present in the biological sample. In general, the
conditions for contacting the agent with the biological sample are
conditions known by those of ordinary skill in the art to
facilitate a specific interaction between a molecule and its
cognate (e. g., a protein and its receptor cognate, an antibody and
its protein antigen cognate, a nucleic acid and its complementary
sequence cognate) in a biological sample. Optimal conditions for
facilitating a specific interaction between a molecule and its
cognate are described in U.S. Pat. No. 5,108,921, issued to Low et
al.
[0365] The diagnostic method of the present invention can be
performed in either or both of in vivo and in vitro. Accordingly,
biological sample can be located in vivo or in vitro in the present
invention. For example, the biological sample can be a tissue in
vivo and the agent specific for the TOPK immunogenic polypeptide
can be used to detect the presence of such molecules in the tissue.
Alternatively, the biological sample can be collected or isolated
in vitro (e. g., a blood sample, tumor biopsy, tissue extract). In
a particularly preferred embodiment, the biological sample can be a
cell-containing sample, more preferably a sample containing tumor
cells collected from a subject to be diagnosed or treated.
[0366] Alternatively, the diagnosis can be done, by a method which
allows direct quantification of antigen-specific T cells by
staining with Fluorescein-labelled HLA multimeric complexes (for
example, Altman, J. D. et al., 1996, Science 274: 94; Altman, J. D.
et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10330;). Staining for
intracellular lymphokines, and interferon-gamma release assays or
ELISPOT assays also has been provided. Tetramer staining,
intracellular lymphokine staining and ELISPOT assays all appear to
be at least 10-fold more sensitive than more conventional assays
(Murali-Krishna, K. et al., 1998, Immunity 8: 177; Lalvani, A. et
al., 1997, J. Exp. Med. 186: 859; Dunbar, P. R. et al., 1998, Curr.
Biol. 8: 413;). Pentamers (e.g., US 2004-209295A), dextramers
(e.g., WO 02/072631), and streptamers (e.g., Nature medicine 6,
631-637 (2002)) may also be used.
[0367] For instance, in some embodiments, the present invention
provides a method for diagnosing or evaluating an immunological
response of a subject administered at least one of TOPK peptides of
the present invention, the method including the steps of:
[0368] (a) contacting an immunogen with immunocompetent cells under
the condition suitable for induction of CTL specific to the
immunogen;
[0369] (b) detecting or determining induction level of the CTL
induced in step (a); and
[0370] (c) correlating the immunological response of the subject
with the CTL induction level.
[0371] In the context of the present invention, the immunogen
preferably includes at least one of (a) a TOPK peptide selected
from among the amino acid sequences of SEQ ID NOs: 2 to 40 and 42
to 84, peptides having such amino acid sequences, and peptides
having in which such amino acid sequences have been modified with
1, 2 or more amino acid substitution(s). In the meantime,
conditions suitable of induction of immunogen specific CTL are well
known in the art. For example, immunocompetent cells may be
cultured in vitro under the presence of immunogen(s) to induce
immunogen specific CTL. In order to induce immunogen specific CTLs,
any stimulating factors may be added to the cell culture. For
example, IL-2 is preferable stimulating factors for the CTL
induction.
[0372] In some embodiments, the step of monitoring or evaluating
immunological response of a subject to be treated with peptide
cancer therapy may be performed before, during and/or after the
treatment. In general, during a protocol of cancer therapy,
immunogenic peptides are administered repeatedly to a subject to be
treated. For example, immunogenic peptides may be administered
every week for 3-10 weeks. Accordingly, the immunological response
of the subject can be evaluated or monitored during the cancer
therapy protocol. Alternatively, the step of evaluation or
monitoring of immunological response to the cancer therapy may at
the completion of the therapy protocol.
[0373] According to the present invention, enhanced induction of
immunogen specific CTL as compared with a control indicates that
the subject to be evaluated or diagnosed immunologically responded
to the immunogen(s) that has/have been administered. Suitable
controls for evaluating the immunological response may include, for
example, a CTL induction level when the immunocompetent cells are
contacted with no peptide, or control peptide(s) having amino acid
sequences other than any TOPK peptides. (e.g. random amino acid
sequence). In a preferred embodiment, the immunological response of
the subject is evaluated in a sequence specific manner, by
comparison with an immunological response between each immunogen
administered to the subject. In particular, even when a mixture of
some kinds of TOPK peptides is administered to the subject,
immunological response might vary depending on the peptides. In
that case, by comparison of the immunological response between each
peptide, peptides to which the subject show higher response can be
identified.
XII. ANTIBODIES
[0374] The present invention further provides antibodies that bind
to peptides of the present invention. Preferred antibodies
specifically bind to peptides of the present invention and will not
bind (or will bind weakly) to other peptides. Alternatively,
antibodies may bind to peptides of the invention as well as the
homologs thereof. Antibodies against peptides of the invention can
find use in cancer diagnostic and prognostic assays, as well as
imaging methodologies. Similarly, such antibodies can find use in
the treatment, diagnosis, and/or prognosis of other cancers, to the
extent TOPK is also expressed or over-expressed in a cancer
patient. Moreover, intracellularly expressed antibodies (e.g.,
single chain antibodies) may therapeutically find use in treating
cancers in which the expression of TOPK is involved, example of
which include, but are not limited to, AML, bladder cancer, breast
cancer, cervical cancer, cholangiocellular carcinoma, colorectal
cancer, diffuse-type gastric cancer, NSCLC, lymphoma, osteosarcoma,
prostate cancer, renal carcinoma, SCLC and soft tissue tumor.
[0375] The present invention also provides various immunological
assays for the detection and/or quantification of the TOPK protein
(SEQ ID NO: 86) or fragments thereof, including polypeptides having
amino acid sequences selected from the group consisting of SEQ ID
NOs: 2 to 40 and 42 to 84. Such assays may include one or more
anti-TOPK antibodies capable of recognizing and binding a TOPK
protein or fragments thereof, as appropriate. In the context of the
present invention, anti-TOPK antibodies binding to TOPK polypeptide
preferably recognize polypeptide having amino acid sequences
selected from the group consisting of SEQ ID NOs: 2 to 40 and 42 to
84, preferably to the exclusion of other peptides. The binding
specificity of antibody can be confirmed by means of an inhibition
test. That is, when the binding between an antibody to be analyzed
and full-length of TOPK polypeptide is inhibited under presence of
any fragment polypeptide having an amino acid sequence of SEQ ID
NOs: 2 to 40 and 42 to 84, it is deemed to specifically bind the
fragment. In the context of the present invention, such
immunological assays are performed within various immunological
assay formats well known in the art, including but not limited to,
various types of radioimmunoassays, immuno-chromatograph technique,
enzyme-linked immunosorbent assays (ELISA), enzyme-linked
immunofluorescent assays (ELIFA), and the like.
[0376] Related immunological but non-antibody assays of the
invention may also include T cell immunogenicity assays (inhibitory
or stimulatory) as well as MHC binding assays. In addition, the
present invention contemplates immunological imaging methods
capable of detecting cancers expressing TOPK, example of which
include, but are not limited to, radioscintigraphic imaging methods
using labeled antibodies of the present invention. Such assays find
clinical use in the detection, monitoring, and prognosis of TOPK
expressing cancers, examples of which include, but are not limited
to, AML, bladder cancer, breast cancer, cervical cancer,
cholangiocellular carcinoma, colorectal cancer, diffuse-type
gastric cancer, NSCLC, lymphoma, osteosarcoma, prostate cancer,
renal carcinoma. SCLC and soft tissue tumor.
[0377] The present invention also provides antibodies that bind to
the peptides of the invention. An antibody of the invention can be
used in any form, for example as a monoclonal or polyclonal
antibody, and may further include antiserum obtained by immunizing
an animal such as a rabbit with the peptide of the invention, all
classes of polyclonal and monoclonal antibodies, human antibodies
and humanized antibodies produced by genetic recombination.
[0378] A peptide of the invention used as an antigen to obtain an
antibody may be derived from any animal species, but is preferably
derived from a mammal such as a human, mouse, or rat, more
preferably from a human. A human-derived peptide may be obtained
from the nucleotide or amino acid sequences disclosed herein.
[0379] According to the present invention, complete and partial
peptides of a protein may serve as immunization antigens. Examples
of suitable partial peptides include, for example, the amino
(N)-terminal or carboxy (C)-terminal fragment of a peptide of the
present invention.
[0380] Herein, an antibody is defined as a protein that reacts with
either the full length or a fragment of a TOPK peptide. In a
preferred embodiment, an antibody of the present invention can
recognize fragment peptides of TOPK having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2 to 40 and 42 to
84. Methods for synthesizing oligopeptide are well known in the
arts. After the synthesis, peptides may be optionally purified
prior to use as immunogen. In the context of the present invention,
the oligopeptide (e.g., 9- or 10mer) may be conjugated or linked
with carriers to enhance the immunogenicity. Keyhole-limpet
hemocyanin (KLH) is well known as the carrier. Method for
conjugating KLH and peptide are also well known in the arts.
[0381] Alternatively, a gene encoding a peptide of the invention or
fragment thereof may be inserted into a known expression vector,
which is then used to transform a host cell as described herein.
The desired peptide or fragment thereof may be recovered from the
outside or inside of host cells by any standard method, and may
subsequently be used as an antigen. Alternatively, whole cells
expressing the peptide or their lysates or a chemically synthesized
peptide may be used as the antigen.
[0382] Any mammalian animal may be immunized with the antigen,
though preferably the compatibility with parental cells used for
cell fusion is taken into account. In general, animals of Rodentia,
Lagomorpha or Primates may be used. Animals of the family Rodentia
include, for example, mouse, rat and hamster. Animals of the family
Lagomorpha include, for example, rabbit. Animals of the Primate
family include, for example, a monkey of Catarrhini (old world
monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon
and chimpanzees.
[0383] Methods for immunizing animals with antigens are known in
the art. Intraperitoneal injection or subcutaneous injection of
antigens is a standard method for the immunization of mammals. More
specifically, antigens may be diluted and suspended in an
appropriate amount of phosphate buffered saline (PBS),
physiological saline, etc. If desired, the antigen suspension may
be mixed with an appropriate amount of a standard adjuvant, such as
Freund's complete adjuvant, made into emulsion and then
administered to mammalian animals. Preferably, it is followed by
several administrations of antigen mixed with an appropriately
amount of Freund's incomplete adjuvant every 4 to 21 days. An
appropriate carrier may also be used for immunization. After
immunization as above, serum may be examined by a standard method
for an increase in the amount of desired antibodies.
[0384] Polyclonal antibodies against the peptides of the present
invention may be prepared by collecting blood from the immunized
mammal examined for the increase of desired antibodies in the
serum, and by separating serum from the blood by any conventional
method. Polyclonal antibodies may include serum containing the
polyclonal antibodies, as well as the fraction containing the
polyclonal antibodies may be isolated from the serum.
Immunoglobulin G or M can be prepared from a fraction which
recognizes only the peptide of the present invention using, for
example, an affinity column coupled with the peptide of the present
invention, and further purifying this fraction using protein A or
protein G column.
[0385] To prepare monoclonal antibodies for use in the context of
the present invention, immune cells are collected from the mammal
immunized with the antigen and checked for the increased level of
desired antibodies in the serum as described above, and are
subjected to cell fusion. The immune cells used for cell fusion may
preferably be obtained from spleen. Other preferred parental cells
to be fused with the above immunocyte include, for example, myeloma
cells of mammalians, and more preferably myeloma cells having an
acquired property for the selection of fused cells by drugs.
[0386] The above immunocyte and myeloma cells can be fused
according to known methods, for example, the method of Milstein et
al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).
[0387] Resulting hybridomas obtained by cell fusion may be selected
by cultivating them in a standard selection medium, such as HAT
medium (hypoxanthine, aminopterin and thymidine containing medium).
The cell culture is typically continued in the HAT medium for
several days to several weeks, the time being sufficient to allow
all the other cells, with the exception of the desired hybridoma
(non-fused cells), to die. Then, the standard limiting dilution may
be performed to screen and clone a hybridoma cell producing the
desired antibody.
[0388] In addition to the above method, wherein a non-human animal
is immunized with an antigen for preparing hybridoma, human
lymphocytes such as those infected by EB virus may be immunized
with a peptide, peptide expressing cells or their lysates in vitro.
Then, the immunized lymphocytes may be fused with human-derived
myeloma cells that are capable of indefinitely dividing, such as
U266, to yield a hybridoma producing a desired human antibody that
is able to bind to the peptide can be obtained (Unexamined
Published Japanese Patent Application No. Sho 63-17688).
[0389] The obtained hybridomas may then be subsequently
transplanted into the abdominal cavity of a mouse and the ascites
extracted. The obtained monoclonal antibodies can be purified by,
for example, ammonium sulfate precipitation, a protein A or protein
G column, DEAE ion exchange chromatography or an affinity column to
which the peptide of the present invention is coupled. An antibody
of the present invention can be used not only for purification and
detection of a peptide of the present invention, but also as a
candidate for agonists and antagonists of a peptide of the present
invention. Alternatively, an immune cell, such as an immunized
lymphocyte, producing antibodies may be immortalized by an oncogene
and used for preparing monoclonal antibodies.
[0390] Monoclonal antibodies thus obtained can be also
recombinantly prepared using genetic engineering techniques (see,
for example, Borrebaeck and Larrick, Therapeutic Monoclonal
Antibodies, published in the United Kingdom by MacMillan Publishers
LTD (1990)). For example, a DNA encoding an antibody may be cloned
from an immune cell, such as a hybridoma or an immunized lymphocyte
producing the antibody, inserted into an appropriate vector, and
introduced into host cells to prepare a recombinant antibody. The
present invention also provides for recombinant antibodies prepared
as described above.
[0391] An antibody of the present invention may be a fragment of an
antibody or modified antibody, so long as it binds to one or more
of the peptides of the invention. For instance, the antibody
fragment may be Fab, F(ab').sub.2, Fv or single chain Fv (scFv), in
which Fv fragments from H and L chains are ligated by an
appropriate linker (Huston et al., Proc Natl Acad Sci USA 85:
5879-83 (1988)). More specifically, an antibody fragment may be
generated by treating an antibody with an enzyme, such as papain or
pepsin. Alternatively, a gene encoding the antibody fragment may be
constructed, inserted into an expression vector and expressed in an
appropriate host cell (see, for example, Co et al., J Immunol 152:
2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96
(1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989);
Lanmoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al.,
Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends
Biotechnol 9: 132-7 (1991)).
[0392] An antibody may be modified by conjugation with a variety of
molecules, such as polyethylene glycol (PEG). The present invention
provides for such modified antibodies. The modified antibody can be
obtained by chemically modifying an antibody. These modification
methods are conventional in the field.
[0393] Alternatively, an antibody of the present invention may be
obtained as a chimeric antibody, between a variable region derived
from nonhuman antibody and the constant region derived from human
antibody, or as a humanized antibody, including the complementarity
determining region (CDR) derived from nonhuman antibody, the frame
work region (FR) and the constant region derived from human
antibody. Such antibodies can be prepared according to known
technology. Humanization can be performed by substituting rodent
CDRs or CDR sequences for the corresponding sequences of a human
antibody (see, e.g., Verhoeyen et al., Science 239:1534-1536
(1988)). Accordingly, such humanized antibodies are chimeric
antibodies, wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species.
[0394] Fully human antibodies including human variable regions in
addition to human framework and constant regions can also be used.
Such antibodies can be produced using various techniques known in
the art. For example, in vitro methods involve use of recombinant
libraries of human antibody fragments displayed on bacteriophage
(e.g., Hoogenboom & Winter. J. Mol. Biol. 227:381 (1991).
Similarly, human antibodies can be made by introducing of human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. This approach is described, e.g., in U.S.
Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016.
[0395] Antibodies obtained as above may be purified to homogeneity.
For example, the separation and purification of the antibody can be
performed according to the separation and purification methods used
for general proteins. For example, the antibody may be separated
and isolated by the appropriately selected and combined use of
column chromatographies, such as affinity chromatography, filter,
ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel
electrophoresis and isoelectric focusing (Antibodies: A Laboratory
Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory
(1988)), but are not limited thereto. A protein A column and
protein G column can be used as the affinity column. Exemplary
protein A columns to be used include, for example, Hyper D, POROS
and Sepharose F.F. (Pharmacia).
[0396] Examples of suitable chromatography techniques, with the
exception of affinity chromatography include, for example,
ion-exchange chromatography, hydrophobic chromatography, gel
filtration, reverse phase chromatography, adsorption chromatography
and the like (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press (1996)). The
chromatographic procedures can be carried out by liquid-phase
chromatography, such as HPLC and FPLC.
[0397] For example, measurement of absorbance, enzyme-linked
immunosorbent assay (ELISA), enzyme immunoassay (EIA),
radioimmunoassay (RIA) and/or immunofluorescence may be used to
measure the antigen binding activity of the antibody of the
invention. In ELISA, the antibody of the present invention is
immobilized on a plate, a peptide of the invention is applied to
the plate, and then a sample containing a desired antibody, such as
culture supernatant of antibody producing cells or purified
antibodies, is applied. Then, a secondary antibody that recognizes
the primary antibody and is labeled with an enzyme, such as
alkaline phosphatase, is applied, and the plate is incubated. Next,
after washing, an enzyme substrate, such as p-nitrophenyl
phosphate, is added to the plate, and the absorbance is measured to
evaluate the antigen binding activity of the sample. A fragment of
the peptide, such as a C-terminal or N-terminal fragment, may be
used as the antigen to evaluate the binding activity of the
antibody. BIAcore (Pharmacia) may be used to evaluate the activity
of the antibody according to the present invention.
[0398] The above methods allow for the detection or measurement of
a peptide of the invention, by exposing an antibody of the
invention to a sample presumed to contain a peptide of the
invention, and detecting or measuring the immune complex formed by
the antibody and the peptide.
[0399] Because the method of detection or measurement of the
peptide according to the invention can specifically detect or
measure a peptide, the method can find use in a variety of
experiments in which the peptide is used.
XIII. VECTORS AND HOST CELLS
[0400] The present invention also provides for vectors and host
cells into which a nucleotide encoding a peptide of the present
invention is introduced. A vector of the present invention finds
utility as a carrier of nucleotides, especially a DNA, of the
present invention in host cell, to express a peptide of the present
invention, or to administer a nucleotide of the present invention
for gene therapy.
[0401] When E. coli is selected as the host cell and the vector is
amplified and produced in a large amount in E. coli (e.g., JM109,
DH5 alpha, HB101 or XL1Blue), the vector should have an "ori" to
suitable for amplification in E. coli and a marker gene suited for
selecting transformed E. coli (e.g., a drug-resistance gene
selected by a drug such as ampicillin, tetracycline, kanamycin,
chloramphenicol or the like). For example, M13-series vectors,
pUC-series vectors, pBR322, pBluescript, pCR-Script, etc., can be
used. In addition, pGEM-T, pDIRECT and pT7 can also be used for
subcloning and extracting cDNA as well as the vectors described
above. When a vector is used to produce the protein of the present
invention, an expression vector can find use. For example, an
expression vector to be expressed in E. coli should have the above
characteristics to be amplified in E. coli. When E. coli, such as
JM109, DH5 alpha, HB101 or XL1 Blue, are used as a host cell, the
vector should have a promoter, for example, lacZ promoter (Ward et
al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1992)), araB
promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter
or the like, that can efficiently express the desired gene in E.
coli. In that respect, pGEX-5X-1 (Pharmacia), "QIAexpress system"
(Qiagen), pEGFP and pET (in this case, the host is preferably BL21
which expresses T7 RNA polymerase), for example, can be used
instead of the above vectors. Additionally, the vector may also
contain a signal sequence for peptide secretion. An exemplary
signal sequence that directs the peptide to be secreted to the
periplasm of the E. coli is the pelB signal sequence (Lei et al., J
Bacteriol 169: 4379 (1987)). Means for introducing of the vectors
into the target host cells include, for example, the calcium
chloride method, and the electroporation method.
[0402] In addition to E. coli, for example, expression vectors
derived from mammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS
(Nucleic Acids Res 18(17): 5322 (1990)), pEF, pCDM8), expression
vectors derived from insect cells (for example, "Bac-to-BAC
baculovirus expression system" (GIBCO BRL), pBacPAK8), expression
vectors derived from plants (e.g., pMH1, pMH2), expression vectors
derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression
vectors derived from retroviruses (e.g., pZIpneo), expression
vector derived from yeast (e.g., "Pichia Expression Kit"
(Invitrogen), pNV11, SP-Q01) and expression vectors derived from
Bacillus subtilis (e.g., pP608, pKTH50) can be used for producing
the polypeptide of the present invention.
[0403] In order to express the vector in animal cells, such as CHO,
COS or NIH3T3 cells, the vector should have a promoter necessary
for expression in such cells, for example, the SV40 promoter
(Mulligan et al., Nature 277: 108 (1979)), the MMLV-LTR promoter,
the EF1 alpha promoter (Mizushima et al., Nucleic Acids Res 18:
5322 (1990)), the CMV promoter and the like, and preferably a
marker gene for selecting transformants (for example, a drug
resistance gene selected by a drug (e.g., neomycin, G418)).
Examples of known vectors with these characteristics include, for
example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
[0404] Hereinafter, the present invention is described in more
detail with reference to the Examples. However, while the following
materials, methods and examples may serve to assist one of ordinary
skill in making and using certain embodiments of the present
invention, there are only intended to illustrate aspects of the
present invention and thus in no way to limit the scope of the
present invention. As one of ordinary skill in the art will readily
recognize, methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention.
Examples
Materials and Methods
[0405] Cell Lines
[0406] TISI, HLA-A*2402-positive B-lymphoblastoid cell line, was
purchased from IHWG Cell and Gene Bank (Seattle, Wash.). T2,
HLA-A*0201-positive B-lymphoblastoid cell line, and COS7, African
green monkey kidney cell line, were purchased from ATCC.
[0407] Candidate Selection of Peptides Derived from TOPK
[0408] 9-mer and 10-mer peptides derived from TOPK (GenBank
Accession No. NM_018492; for example, SEQ ID No: 85) that bind to
either or both of HLA-A*2402 and HLA-A*0201 molecule were predicted
using "NetMHC3.0" binding prediction server
(http://www.cbs.dtu.dk/services/NetMHC/) (Buus et al., Tissue
Antigens. 2003 November, 62(5):378-84; Nielsen et al., Protein Sci.
2003 May, 12(5):1007-17, Bioinformatics. 2004 Jun.
12:20(9):1388-97). These peptides were synthesized by Biosynthesis
(Lewisville, Tex.) according to a standard solid phase synthesis
method and purified by reversed phase high performance liquid
chromatography (HPLC). The purity (>90%) and the identity of the
peptides were determined by analytical HPLC and mass spectrometry
analysis, respectively. Peptides were dissolved in
dimethylsulfoxide at 20 mg/ml and stored at -80 degrees C.
[0409] In Vitro CTL Induction
[0410] Monocyte-derived dendritic cells (DCs) were used as
antigen-presenting cells to induce cytotoxic T lymphocyte (CTL)
responses against peptides presented on human leukocyte antigen
(HLA). DCs were generated in vitro as described elsewhere (Nakahara
S et al., Cancer Res 2003 Jul. 15, 63(14): 4112-8). Specifically,
peripheral blood mononuclear cells isolated from a normal volunteer
(HLA-A*2402 positive or HLA-A*0201 positive) by Ficoll-Paque plus
(Pharmacia) solution were separated by adherence to a plastic
tissue culture dish (Becton Dickinson) so as to enrich them as the
monocyte fraction. The monocyte-enriched population was cultured in
the presence of 1000 U/ml of granulocyte-macrophage
colony-stimulating factor (R&D System) and 1000 U/ml of
interleukin (IL)-4 (R&D System) in AIM-V Medium (Invitrogen)
containing 2% heat-inactivated autologous serum (AS). After 7 days
of culture, the cytokine-induced DCs were pulsed with 20 micro-g/ml
of each of the synthesized peptides in the presence of 3 micro g/ml
of beta 2-microglobulin for 3 hr at 37 degrees C. in AIM-V Medium.
The generated cells appeared to express DC-associated molecules,
such as CD80, CD83, CD86 and HLA class II, on their cell surfaces
(data not shown). These peptide-pulsed DCs were then inactivated by
X ray-irradiated (20 Gy) and mixed at a 1:20 ratio with autologous
CD8.sup.+ T cells, obtained by positive selection with CD8 Positive
Isolation Kit (Dynal). These cultures were set up in 48-well plates
(Corning); each well contained 1.5.times.10.sup.4 peptide-pulsed
DCs, 3.times.10.sup.5 CD8.sup.+ T cells and 10 ng/ml of IL-7
(R&D System) in 0.5 ml of AIM-V/2% AS medium. Three days later,
these cultures were supplemented with IL-2 (CHIRON) to a final
concentration of 20 IU/ml. On day 7 and 14, the T cells were
further stimulated with the autologous peptide-pulsed DCs. The DCs
were prepared each time by the same way described above. CTLs were
tested against peptide-pulsed TISI cells or T2 cells after the 3rd
round of peptide stimulation on day 21 (Tanaka H et al., Br J
Cancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001
Apr. 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec.
15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5):
411-9; Watanabe T et al., Cancer Sci 2005 August, 96(8):
498-506).
[0411] CTL Expansion Procedure
[0412] CTLs were expanded in culture using the method similar to
the one described by Riddell et al. (Walter E A et al., N Engl J
Med 1995 Oct. 19, 333(16): 1038-44; Riddell S R et al., Nat Med
1996 February, 2(2): 216-23). A total of 5.times.10.sup.4 CTLs were
suspended in 25 ml of AIM-V/5% AS medium with 2 kinds of human
B-lymphoblastoid cell lines, inactivated by Mitomycin C, in the
presence of 40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen).
One day after initiating the cultures, 120 IU/ml of IL-2 were added
to the cultures. The cultures were fed with fresh AIM-V/5% AS
medium containing 30 IU/ml of IL-2 on days 5, 8 and 11 (Tanaka H et
al., Br J Cancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br J
Cancer 2001 Apr. 20, 84(8): 1052-7; Uchida N et aL, Clin Cancer Res
2004 Dec. 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May,
97(5): 411-9; Watanabe T et al., Cancer Sci 2005 August 96(8):
498-506).
[0413] Establishment of CTL Clones
[0414] The dilutions were made to have 0.3, 1, and 3 CTLs/well in
96 round-bottomed micro titer plate (Nalge Nunc International).
CTLs were cultured with 1.times.10.sup.4 cells/well of 2 kinds of
human B-lymphoblastoid cell lines, 30 ng/ml of anti-CD3 antibody,
and 125 U/ml of IL-2 in a total of 150 micro-1/well of AIM-V Medium
containing 5% AS. 50 micro-l/well of IL-2 were added to the medium
10 days later to reach a final concentration of 125 U/ml IL-2. CTL
activity was tested on the 14th day, and CTL clones were expanded
using the same method as described above (Uchida N et al., Clin
Cancer Res 2004 Dec. 15, 10(24): 8577-86; Suda T et al., Cancer Sci
2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 August,
96(8): 498-506).
[0415] Specific CTL Activity
[0416] To examine specific CTL activity, IFN-gamma ELISPOT assay
and IFN-gamma ELISA were performed. Peptide-pulsed TISI cells or T2
cells (1.times.10.sup.4/well) were prepared as stimulator cells.
Cultured cells in 48-well plate. CTL lines and CTL clones were used
as responder cells. IFN-gamma ELISPOT assay and IFN-gamma ELISA
were performed under the manufacturer's procedure.
[0417] Establishment of the Cells Forcibly Expressing Either or
Both of the Target Gene and HLA-A24 or HLA-A2
[0418] The cDNA encoding an open reading frame of target genes.
HLA-A*2402 or HLA-A*0201 was amplified by PCR. The PCR-amplified
product was cloned into expression vector. The plasmids were
transfected into COS7, which is the target genes-null,
HLA-A*0201-null and HLA-A*2402-null cell line, using lipofectamine
2000 (Invitrogen) according to the manufacturer's procedure. After
2 days from transfection, the transfected cells were harvested with
versene (Invitrogen) and used as the stimulator cells
(5.times.10.sup.4 cells/well) for CTL activity assay.
[0419] CTL Ability to Recognize the Target Cell Line that
Endogenously Expressed TOPK and HLA-A*2402 or HLA-A*0201
[0420] The CTL clone was examined for its ability to recognize the
target cell that endogenously expressed TOPK and HLA-A*2402 or
HLA-A*0201. Established CTL clone was cultured with target cell
lines (5.times.10.sup.4/well) for two overnight. After incubation,
IFN-gamma in the culture media was measured by ELISA. IFN-gamma
ELISA was performed under the manufacturer's procedure.
[0421] Result
[0422] Enhanced TOPK Expression in Cancers
[0423] The wide gene expression profile data obtained from various
cancers using cDNA-microarray revealed that TOPK (GenBank Accession
No. NM_018492; for example, SEQ ID No: 85) expression was
specifically elevated in cancer tissues as compared with
corresponding normal tissue. TOPK expression was validly elevated
in 1 out of 15 AML, 15 out of 18 bladder cancers, 36 out of 40
breast cancers, 2 out of 6 cervical cancers, 6 out of 6
cholangiocellular carcinoma, 2 out of 6 colorectal cancers, 1 out
of 1 diffuse-type gastric cancer, 5 out of 5 NSCLC, 1 out of 2
lymphomas, 7 out of 11 osteosarcoma, 12 out of 19 prostate cancers,
3 out of 12 renal carcinomas, 14 out of 14 SCLCs and 15 out of 29
soft tissue tumors (Table 1).
TABLE-US-00001 TABLE 1 Ratio of cases observed up-regulation of
TOPK in cancerous tissues as compared with normal corresponding
tissues. Cancer/Tumor Ratio AML 1/15 Bladder cancer 15/18 Breast
cancer 36/40 Cervical cancer 2/6 Cholangiocellular carcinoma 6/6
Colorectal cancer 2/6 Diffuse-type gastric cancer 1/1 NSCLC 5/5
Lymphoma 1/2 Osteosarcoma 7/11 Prostate cancer 12/19 Renal
carcinoma 3/12 SCLC 14/14 Soft tissue tumor 15/29
[0424] Prediction of HLA-A24 Binding Peptides Derived from TOPK
[0425] Table 2a and 2b show the HLA-A24 binding of 9mer and 10mer
peptides of TOPK in the order of high binding affinity. A total of
40) peptides having potential HLA-A24 binding ability were selected
and examined to determine the epitope peptides.
TABLE-US-00002 TABLE 2a HLA-A24 binding 9mer peptides derived from
TOPK Start amino SEQ ID Position acid sequence Kd (mM) NO 289
SYQKVIELF 21 1 230 IFAFGLTLW 363 2 130 RYKASQDPF 451 3 237
LWEMMTLSI 1351 4 155 KYLHQEKKL 1906 5 232 AFGLTLWEM 3946 6 174
VIKGDFETI 4496 7 73 HYRSVYQKR 4663 8 235 LILWEMMTL 4781 9 19
SVLCSTPTI 6522 10 205 CYIGTEPWK 7254 11 77 VYQKRLMDE 8604 12 270
AYYAALGTR 8621 13 58 HSPWAVKKI 9096 14 81 RLMDEAKIL 12527 15 278
RPPINMEEL 19706 16 183 KICDVGVSL 25266 17 227 KADIFAFGL 25408 18 13
LSEKKKSVL 26380 19 146 VALNMARGL 26693 20 140 AAIILKVAL 28349 21
103 FTEANDGSL 29275 22 105 EANDGSLCL 29821 73 118 GGEKSLNDL 35171
24
TABLE-US-00003 TABLE 2b HLA-A24 binding 10mer peptides derived from
TOPK Start amino Position acid sequence Kd (nM) SEQ ID NO 31
ASPFMQKLGF 4764 25 155 KYLHQEKKLL 8099 26 288 ESYQKVIELF 9466 27
289 SYQKVIELFS 9631 28 130 RYKASQDPFP 9917 29 47 YLMKRSPRGL 10978
30 73 HYRSVYQKRL 11919 31 102 AFTEANDGSL 14375 32 39 GFGTGVNVYL
21925 33 4 ISNFKTPSKL 21974 34 77 VYQKRLMDEA 23521 35 241
MTLSIPHINL 27049 36 12 KLSEKKKSVL 28153 37 148 LNMARGLKYL 30397 38
145 KVALNMARGL 32052 39 114 AMEYGGEKSL: 32705 40 Start position
indicates the number of amino acid residue from the N-terminus of
TOPK. Dissociation constant [Kd(nM)] is derived from ''NetMHC
3.0''
[0426] Prediction of HLA-A02 Binding Peptides Derived from TOPK
[0427] Table 3a and 3b show the HLA-A02 binding 9mer and 10mer
peptides of TOPK respectively in the order of high binding
affinity. A total of 44 peptides with potential HLA-A02 binding
ability were selected and examined to determine the epitope
peptides.
TABLE-US-00004 TABLE 3a HLA-A02 binding 9mer peptides derived from
TOPK Start amino Position acid sequence Kd (nM) SEQ ID NO 55
GLSHSPWAV 13 41 240 MMTLSIPHI 37 42 34 FMQKLGFGT 76 43 236
TLWEMMTLS 150 44 19 SVLCSTPTI 230 45 134 SQDPFPAAI 238 46 183
KICDVGVSL 415 47 81 RLMDEAKIL 470 48 149 NMARGLKYL 524 49 235
LTLWEMMTL 648 50 12 KLSEKKKSV 775 51 277 KADIFAFGL 1542 52 285
ELDESYQKV 1902 53 47 YLMKRSPRG 2476 54 310 SAAHIVEAL 3199 55 132
KASQDPFPA 3496 56 242 TLSIPHINL 3753 57 156 YLHQEKKLL 4077 58 138
FPAAIILKV 4228 59 142 IILKVALNM 4330 60
TABLE-US-00005 TABLE 3b HLA-A02 binding 10mer peptides derived from
TOPK Start amino Position acid sequence Kd (nM) SEQ ID NO 190
SLPLDENMTV 30 61 236 TLWEMMTLSI 32 62 231 FAFGLTLWEM 41 63 47
YLMKRSPRGL 64 64 234 GLTIWEMMTL 74 65 239 EMMTLSIPHI 93 66 290
YQKVIELFSV 101 67 37 KLGFGTGVNV 192 68 20 VLCSTPTINI 290 69 241
MTLSIPHINL 310 70 272 YAALGTRPPI 1347 71 88 ILKSLHHPNI 1656 72 81
RLMDEAKILK 1720 73 313 HIVEALETDV 2345 74 54 RGLSHSPWAV 2364 75 142
IILKVALNMA 2428 76 35 MQKLGFGTGV 2432 77 110 SLCLAMEYGG 3236 78 223
VITDKADIFA 3422 79 274 ALGTRPPINM 3575 80 173 VVIKGDFETI 3955 81
141 AIILKVALNM 4247 87 292 KVIELFSVCT 4637 83 180 ETIKICDVGV 4911
84 Start position indicates the number of amino acid residue from
the N-terminus of TOPK. Dissociation constant [Kd(nM)] is derived
from ''NetMHC 3.0''.
[0428] CTL Induction with the Predicted Peptides from TOPK
Restricted with HLA-A*2402
[0429] CTLs for those peptides derived from TOPK were generated
according to the protocols as described in "Materials and Methods".
Peptide specific CTL activity was detected by IFN-gamma ELISPOT
assay (FIG. 1). Well number #8 with TOPK-A24-9-230 (SEQ ID NO: 2)
(a), #3 with TOPK-A24-9-130 (SEQ ID NO: 3) (b), #3 with
TOPK-A24-9-232 (SEQ ID NO: 6) (c), #2 with TOPK-A24-10-288 (SEQ ID
NO: 27) (d) and #4 with TOPK-A24-10-289 (SEQ ID NO: 28) (e)
demonstrated potent IFN-gamma production as compared to the control
wells. On the other hand, no specific CTL activity was detected by
stimulation with other peptides shown in Table 2a and 2b, despite
those peptides had possible binding activity with HLA-A*2402. As is
typical of negative data, no specific IFN-gamma production was
observed from the CTL stimulated with TOPK-A24-9-289 (SEQ ID NO: 1)
(f). Taken together, these results suggest that the 5 selected
peptides derived from TOPK could induce potent CTLs.
[0430] CTL Induction with the Predicted Peptides from TOPK
Restricted with HLA-A*0201
[0431] Peptide specific CTL activity was detected by IFN-gamma
ELISPOT assay (FIG. 2). Well number #7 with TOPK-A02-9-240 (SEQ ID
NO: 42) (a), #4 with TOPK-A02-9-19 (SEQ ID NO: 45) (b). #2 with
TOPK-A02-9-183 (SEQ ID NO: 47) (c), #8 with TOPK-A02-9-235 (SEQ ID
NO: 50) (d), #4 with TOPK-A02-9-12 (SEQ ID NO: 51) (e), #3 with
TOPK-A02-9-285 (SEQ ID NO: 53) (f). #3 with TOPK-A02-9-47 (SEQ ID
NO: 54) (g). #5 with TOPK-A02-10-236 (SEQ ID NO: 62) (h), #3 with
TOPK-A02-10-231 (SEQ ID NO: 63) (i), #8 with TOPK-A02-10-47 (SEQ ID
NO: 64) (j). #1 with TOPK-A02-10-239 (SEQ ID NO: 66) (k), #1 with
TOPK-A02-10-272 (SEQ ID NO: 71) (l), #4 with TOPK-A02-10-88 (SEQ ID
NO: 72) (m) and #4 with TOPK-A02-10-142 (SEQ ID NO: 76) (n)
demonstrated potent IFN-gamma production as compared to the control
wells. On the other hand, no specific CTL activity was detected by
stimulation with other peptides shown in Table 3a and 3b, despite
those peptides had possible binding activity with HLA-A*0201. As is
typical of negative data, no specific IFN-gamma production was
observed from the CTL stimulated with TOPK-A02-9-55 (SEQ ID NO: 41)
(o). Taken together, these results suggest that the 14 selected
peptides derived from TOPK could induce potent CTLs.
[0432] Establishment of CTL Line and Clone Against TOPK Derived
Peptide
[0433] The cells in the well number #8 with TOPK-A24-9-230 (SEQ ID
NO: 2) (a), #3 with TOPK-A24-9-130 (SEQ ID NO: 3) (b), #3 with
TOPK-A24-9-232 (SEQ ID NO: 6) (c), #2 with TOPK-A24-10-288 (SEQ ID
NO: 27) (d) and #4 with TOPK-A24-10-289 (SEQ ID NO: 28) (e) that
showed peptide specific CTL activity by IFN-gamma ELISPOT assay
were expanded and established the CTL lines. CTL activities of
these CTL lines were measured by IFN-gamma ELISA (FIG. 3). CTL
lines demonstrated potent IFN-gamma production against target cells
pulsed with the corresponding peptide as compared to target cells
without peptide pulse. Furthermore, the CTL clones were established
by limiting dilution from the CTL lines as described in "Materials
and Methods", and IFN-gamma production from the CTL clones against
TISI cells pulsed with corresponding peptide was measured by
IFN-gamma ELISA. Potent IFN-gamma production was observed from the
CTL clones stimulated with TOPK-A24-9-130 (SEQ ID NO: 3) (a),
TOPK-A24-10-288 (SEQ ID NO: 27) (b) and TOPK-A24-10-289 (SEQ ID NO:
28) (c) (FIG. 4).
[0434] The cells in the well number #7 with TOPK-A02-9-240 (SEQ ID
NO: 42) (a), #4 with TOPK-A02-9-19 (SEQ ID NO: 45) (b), #8 with
TOPK-A02-9-235 (SEQ ID NO: 50) (C), #4 with TOPK-A02-9-12 (SEQ ID
NO: 51) (d). #3 with TOPK-A02-9-285 (SEQ ID NO: 53) (e), #3 with
TOPK-A02-9-47 (SEQ ID NO: 54) (f), #5 with TOPK-A02-10-236 (SEQ ID
NO: 62) (g), #3 with TOPK-A02-10-231 (SEQ ID NO: 63) (h), #8 with
TOPK-A02-10-47 (SEQ ID NO: 64) (i), #1 with TOPK-A02-10-239 (SEQ ID
NO: 66) (j) and #4 with TOPK-A02-10-88 (SEQ ID NO: 72) (k) that
showed peptide specific CTL activity by IFN-gamma ELISPOT assay
were expanded and established the CTL lines. The CTL activities of
these CTL lines were measured by IFN-gamma ELISA (FIG. 5). CTL
lines demonstrated potent IFN-gamma production against target cells
pulsed with the corresponding peptide as compared to target cells
without peptide pulse. Furthermore, the CTL clones were established
by limiting dilution from the CTL lines as described in "Materials
and Methods", and IFN-gamma production from the CTL clones against
T2 cells pulsed with corresponding peptide was measured by
IFN-gamma ELISA. Potent IFN-gamma production was observed from the
CTL clones stimulated with TOPK-A02-9-240 (SEQ ID NO: 42) (a) and
TOPK-A02-9-285 (SEQ ID NO: 53) (b) (FIG. 6).
[0435] Specific CTL Activity Against Target Cells Expressing TOPK
and HLA-A*2402 or HLA-A*0201
[0436] The established CTL clone raised against TOPK-A24-10-289
(SEQ ID NO: 28) peptide was examined for the ability to recognize
target cells that express TOPK and HLA-A*2402 molecule. COS7 cells
transfected with both the full length of TOPK and HLA-A*2402 gene
(a specific model for the target cells that express TOPK and
HLA-A*2402 gene) were prepared as a stimulator cells, and COS7
cells transfected with either full length of TOPK or HLA-A*2402
were used as the controls. In FIG. 7, the CTL clone stimulated with
TOPK-A24-10-289 (SEQ ID NO: 28) showed potent CTL activity against
COS7 cells expressing both TOPK and HLA-A*2402. On the other hand,
no significant specific CTL activity was detected against the
controls. Thus, these data clearly demonstrate that TOPK-A24-10-289
(SEQ ID NO: 28) peptide is endogenously processed and expressed on
the target cells with HLA-A*2402 molecule and is recognized by the
CTLs. The established CTL line raised against TOPK-A02-9-240 (SEQ
ID NO: 42) peptide was examined for the ability to recognize target
cells that express TOPK and HLA-A*0201 molecule. COS7 cells
transfected with both the full length of TOPK and HLA-A*0201 gene
(a specific model for the target cells that express TOPK and
HLA-A0201 gene) were prepared as a stimulator cells, and COS7 cells
transfected with either full length of TOPK or HLA-A*0201 were used
as the controls. In FIG. 8, the CTL line stimulated with
TOPK-A02-9-240 (SEQ ID NO: 42) showed potent CTL activity against
COS7 cells expressing both TOPK and HLA-A*0201. On the other hand,
no significant specific CTL activity was detected against the
controls. Thus, these data clearly demonstrate that TOPK-A02-9-240
(SEQ ID NO: 42) peptide is endogenously processed and expressed on
the target cells with HLA-A*0201 molecule and is recognized by the
CTLs. These results indicate that these peptides derived from TOPK
may be available to apply the cancer vaccines for patients with
TOPK expressing tumors.
[0437] Homology Analysis of Antigen Peptides
[0438] The CTLs stimulated with TOPK-A24-9-230 (SEQ ID NO: 2),
TOPK-A24-9-130 (SEQ ID NO: 3), TOPK-A24-9-232 (SEQ ID NO: 6),
TOPK-A24-10-288 (SEQ ID NO: 27), TOPK-A24-10-289 (SEQ ID NO: 28),
TOPK-A02-9-240 (SEQ ID NO: 42). TOPK-A02-9-19 (SEQ ID NO: 45),
TOPK-A02-9-183 (SEQ ID NO: 47), TOPK-A02-9-235 (SEQ ID NO: 50),
TOPK-A02-9-12 (SEQ ID NO: 51), TOPK-A02-9-285 (SEQ ID NO: 53),
TOPK-A02-9-47 (SEQ ID NO: 54), TOPK-A02-10-236 (SEQ ID NO: 62),
TOPK-A02-10-231 (SEQ ID NO: 63), TOPK-A02-10-47 (SEQ ID NO: 64),
TOPK-A02-10-239 (SEQ ID NO: 66), TOPK-A02-10-272 (SEQ ID NO: 71),
TOPK-A02-10-88 (SEQ ID NO: 72) or TOPK-A02-10-142 (SEQ ID NO: 76)
showed significant and specific CTL activity. This result may be
due to the fact that these sequences are homologous to peptide
derived from other molecules that are known to sensitize the human
immune system. To exclude this possibility, homology analyses were
performed for these peptide sequences using as queries the BLAST
algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) which
revealed no sequence with significant homology. The results of
homology analyses indicate that the sequence of TOPK-A24-9-230 (SEQ
ID NO: 2), TOPK-A24-9-130 (SEQ ID NO: 3), TOPK-A24-9-232 (SEQ ID
NO: 6), TOPK-A24-10-288 (SEQ ID NO: 27), TOPK-A24-10-289 (SEQ ID
NO: 28), TOPK-A02-9-240 (SEQ ID NO: 42), TOPK-A02-9-19 (SEQ ID NO:
45), TOPK-A02-9-183 (SEQ ID NO: 47), TOPK-A02-9-235 (SEQ ID NO:
50), TOPK-A02-9-12 (SEQ ID NO: 5), TOPK-A02-9-285 (SEQ ID NO: 53),
TOPK-A02-9-47 (SEQ ID NO: 54), TOPK-A02-10-236 (SEQ ID NO: 62),
TOPK-A02-10-231 (SEQ ID NO: 63), TOPK-A02-10-47 (SEQ ID NO: 64),
TOPK-A02-10-239 (SEQ ID NO: 66), TOPK-A02-10-272 (SEQ ID NO: 71),
TOPK-A02-10-88 (SEQ ID NO: 72) and TOPK-A02-10-142 (SEQ ID NO: 76)
are unique and thus, there is little possibility, to our best
knowledge, that this molecules raise unintended immunologic
response to some unrelated molecule. In conclusion, the novel
HLA-A24 or HLA-A02 epitope peptides derived from TOPK identified
herein may find utility in the field of cancer immunotherapy.
INDUSTRIAL APPLICABILITY
[0439] The present invention provides new TAAs, particularly those
derived from TOPK, that may induce potent and specific anti-tumor
immune responses and thus have applicability to a wide variety of
cancer types. Such TAAs can find use as peptide vaccines against
diseases associated with TOPK, e.g., cancer, more particularly,
acute myeloid leukemia (AML), bladder cancer, breast cancer,
cervical cancer, cholangiocellular carcinoma, colorectal cancer,
diffuse-type gastric cancer, non small cell lung cancer (NSCLC),
lymphoma, osteosarcoma, prostate cancer, renal carcinoma, small
cell lung cancer (SCLC) and soft tissue tumor.
[0440] While the present invention is herein described in detail
and with reference to specific embodiments thereof, it is to be
understood that the foregoing description is exemplary and
explanatory in nature and is intended to illustrate the present
invention and its preferred embodiments. Through routine
experimentation, one skilled in the art will readily recognize that
various changes and modifications can be made therein without
departing from the spirit and scope of the present invention, the
metes and bounds of which are defined by the appended claims.
Sequence CWU 1
1
9019PRTArtificial Sequencea peptide derived from TOPK 1Ser Tyr Gln
Lys Val Ile Glu Leu Phe 1 5 29PRTArtificial Sequencea peptide
derived from TOPK 2Ile Phe Ala Phe Gly Leu Thr Leu Trp 1 5
39PRTArtificial Sequencea peptide derived from TOPK 3Arg Tyr Lys
Ala Ser Gln Asp Pro Phe 1 5 49PRTArtificial Sequencea peptide
derived from TOPK 4Leu Trp Glu Met Met Thr Leu Ser Ile 1 5
59PRTArtificial Sequencea peptide derived from TOPK 5Lys Tyr Leu
His Gln Glu Lys Lys Leu 1 5 69PRTArtificial Sequencea peptide
derived from TOPK 6Ala Phe Gly Leu Thr Leu Trp Glu Met 1 5
79PRTArtificial Sequencea peptide derived from TOPK 7Val Ile Lys
Gly Asp Phe Glu Thr Ile 1 5 89PRTArtificial Sequencea peptide
derived from TOPK 8His Tyr Arg Ser Val Tyr Gln Lys Arg 1 5
99PRTArtificial Sequencea peptide derived from TOPK 9Leu Thr Leu
Trp Glu Met Met Thr Leu 1 5 109PRTArtificial Sequencea peptide
derived from TOPK 10Ser Val Leu Cys Ser Thr Pro Thr Ile 1 5
119PRTArtificial Sequencea peptide derived from TOPK 11Cys Tyr Ile
Gly Thr Glu Pro Trp Lys 1 5 129PRTArtificial Sequencea peptide
derived from TOPK 12Val Tyr Gln Lys Arg Leu Met Asp Glu 1 5
139PRTArtificial Sequencea peptide derived from TOPK 13Ala Tyr Tyr
Ala Ala Leu Gly Thr Arg 1 5 149PRTArtificial Sequencea peptide
derived from TOPK 14His Ser Pro Trp Ala Val Lys Lys Ile 1 5
159PRTArtificial Sequencea peptide derived from TOPK 15Arg Leu Met
Asp Glu Ala Lys Ile Leu 1 5 169PRTArtificial Sequencea peptide
derived from TOPK 16Arg Pro Pro Ile Asn Met Glu Glu Leu 1 5
179PRTArtificial Sequencea peptide derived from TOPK 17Lys Ile Cys
Asp Val Gly Val Ser Leu 1 5 189PRTArtificial Sequencea peptide
derived from TOPK 18Lys Ala Asp Ile Phe Ala Phe Gly Leu 1 5
199PRTArtificial Sequencea peptide derived from TOPK 19Leu Ser Glu
Lys Lys Lys Ser Val Leu 1 5 209PRTArtificial Sequencea peptide
derived from TOPK 20Val Ala Leu Asn Met Ala Arg Gly Leu 1 5
219PRTArtificial Sequencea peptide derived from TOPK 21Ala Ala Ile
Ile Leu Lys Val Ala Leu 1 5 229PRTArtificial Sequencea peptide
derived from TOPK 22Phe Thr Glu Ala Asn Asp Gly Ser Leu 1 5
239PRTArtificial Sequencea peptide derived from TOPK 23Glu Ala Asn
Asp Gly Ser Leu Cys Leu 1 5 249PRTArtificial Sequencea peptide
derived from TOPK 24Gly Gly Glu Lys Ser Leu Asn Asp Leu 1 5
2510PRTArtificial Sequencea peptide derived from TOPK 25Ala Ser Pro
Phe Met Gln Lys Leu Gly Phe 1 5 10 2610PRTArtificial Sequencea
peptide derived from TOPK 26Lys Tyr Leu His Gln Glu Lys Lys Leu Leu
1 5 10 2710PRTArtificial Sequencea peptide derived from TOPK 27Glu
Ser Tyr Gln Lys Val Ile Glu Leu Phe 1 5 10 2810PRTArtificial
Sequencea peptide derived from TOPK 28Ser Tyr Gln Lys Val Ile Glu
Leu Phe Ser 1 5 10 2910PRTArtificial Sequencea peptide derived from
TOPK 29Arg Tyr Lys Ala Ser Gln Asp Pro Phe Pro 1 5 10
3010PRTArtificial Sequencea peptide derived from TOPK 30Tyr Leu Met
Lys Arg Ser Pro Arg Gly Leu 1 5 10 3110PRTArtificial Sequencea
peptide derived from TOPK 31His Tyr Arg Ser Val Tyr Gln Lys Arg Leu
1 5 10 3210PRTArtificial Sequencea peptide derived from TOPK 32Ala
Phe Thr Glu Ala Asn Asp Gly Ser Leu 1 5 10 3310PRTArtificial
Sequencea peptide derived from TOPK 33Gly Phe Gly Thr Gly Val Asn
Val Tyr Leu 1 5 10 3410PRTArtificial Sequencea peptide derived from
TOPK 34Ile Ser Asn Phe Lys Thr Pro Ser Lys Leu 1 5 10
3510PRTArtificial Sequencea peptide derived from TOPK 35Val Tyr Gln
Lys Arg Leu Met Asp Glu Ala 1 5 10 3610PRTArtificial Sequencea
peptide derived from TOPK 36Met Thr Leu Ser Ile Pro His Ile Asn Leu
1 5 10 3710PRTArtificial Sequencea peptide derived from TOPK 37Lys
Leu Ser Glu Lys Lys Lys Ser Val Leu 1 5 10 3810PRTArtificial
Sequencea peptide derived from TOPK 38Leu Asn Met Ala Arg Gly Leu
Lys Tyr Leu 1 5 10 3910PRTArtificial Sequencea peptide derived from
TOPK 39Lys Val Ala Leu Asn Met Ala Arg Gly Leu 1 5 10
4010PRTArtificial Sequencea peptide derived from TOPK 40Ala Met Glu
Tyr Gly Gly Glu Lys Ser Leu 1 5 10 419PRTArtificial Sequencea
peptide derived from TOPK 41Gly Leu Ser His Ser Pro Trp Ala Val 1 5
429PRTArtificial Sequencea peptide derived from TOPK 42Met Met Thr
Leu Ser Ile Pro His Ile 1 5 439PRTArtificial Sequencea peptide
derived from TOPK 43Phe Met Gln Lys Leu Gly Phe Gly Thr 1 5
449PRTArtificial Sequencea peptide derived from TOPK 44Thr Leu Trp
Glu Met Met Thr Leu Ser 1 5 459PRTArtificial Sequencea peptide
derived from TOPK 45Ser Val Leu Cys Ser Thr Pro Thr Ile 1 5
469PRTArtificial Sequencea peptide derived from TOPK 46Ser Gln Asp
Pro Phe Pro Ala Ala Ile 1 5 479PRTArtificial Sequencea peptide
derived from TOPK 47Lys Ile Cys Asp Val Gly Val Ser Leu 1 5
489PRTArtificial Sequencea peptide derived from TOPK 48Arg Leu Met
Asp Glu Ala Lys Ile Leu 1 5 499PRTArtificial Sequencea peptide
derived from TOPK 49Asn Met Ala Arg Gly Leu Lys Tyr Leu 1 5
509PRTArtificial Sequencea peptide derived from TOPK 50Leu Thr Leu
Trp Glu Met Met Thr Leu 1 5 519PRTArtificial Sequencea peptide
derived from TOPK 51Lys Leu Ser Glu Lys Lys Lys Ser Val 1 5
529PRTArtificial Sequencea peptide derived from TOPK 52Lys Ala Asp
Ile Phe Ala Phe Gly Leu 1 5 539PRTArtificial Sequencea peptide
derived from TOPK 53Glu Leu Asp Glu Ser Tyr Gln Lys Val 1 5
549PRTArtificial Sequencea peptide derived from TOPK 54Tyr Leu Met
Lys Arg Ser Pro Arg Gly 1 5 559PRTArtificial Sequencea peptide
derived from TOPK 55Ser Ala Ala His Ile Val Glu Ala Leu 1 5
569PRTArtificial Sequencea peptide derived from TOPK 56Lys Ala Ser
Gln Asp Pro Phe Pro Ala 1 5 579PRTArtificial Sequencea peptide
derived from TOPK 57Thr Leu Ser Ile Pro His Ile Asn Leu 1 5
589PRTArtificial Sequencea peptide derived from TOPK 58Tyr Leu His
Gln Glu Lys Lys Leu Leu 1 5 599PRTArtificial Sequencea peptide
derived from TOPK 59Phe Pro Ala Ala Ile Ile Leu Lys Val 1 5
609PRTArtificial Sequencea peptide derived from TOPK 60Ile Ile Leu
Lys Val Ala Leu Asn Met 1 5 6110PRTArtificial Sequencea peptide
derived from TOPK 61Ser Leu Pro Leu Asp Glu Asn Met Thr Val 1 5 10
6210PRTArtificial Sequencea peptide derived from TOPK 62Thr Leu Trp
Glu Met Met Thr Leu Ser Ile 1 5 10 6310PRTArtificial Sequencea
peptide derived from TOPK 63Phe Ala Phe Gly Leu Thr Leu Trp Glu Met
1 5 10 6410PRTArtificial Sequencea peptide derived from TOPK 64Tyr
Leu Met Lys Arg Ser Pro Arg Gly Leu 1 5 10 6510PRTArtificial
Sequencea peptide derived from TOPK 65Gly Leu Thr Leu Trp Glu Met
Met Thr Leu 1 5 10 6610PRTArtificial Sequencea peptide derived from
TOPK 66Glu Met Met Thr Leu Ser Ile Pro His Ile 1 5 10
6710PRTArtificial Sequencea peptide derived from TOPK 67Tyr Gln Lys
Val Ile Glu Leu Phe Ser Val 1 5 10 6810PRTArtificial Sequencea
peptide derived from TOPK 68Lys Leu Gly Phe Gly Thr Gly Val Asn Val
1 5 10 6910PRTArtificial Sequencea peptide derived from TOPK 69Val
Leu Cys Ser Thr Pro Thr Ile Asn Ile 1 5 10 7010PRTArtificial
Sequencea peptide derived from TOPK 70Met Thr Leu Ser Ile Pro His
Ile Asn Leu 1 5 10 7110PRTArtificial Sequencea peptide derived from
TOPK 71Tyr Ala Ala Leu Gly Thr Arg Pro Pro Ile 1 5 10
7210PRTArtificial Sequencea peptide derived from TOPK 72Ile Leu Lys
Ser Leu His His Pro Asn Ile 1 5 10 7310PRTArtificial Sequencea
peptide derived from TOPK 73Arg Leu Met Asp Glu Ala Lys Ile Leu Lys
1 5 10 7410PRTArtificial Sequencea peptide derived from TOPK 74His
Ile Val Glu Ala Leu Glu Thr Asp Val 1 5 10 7510PRTArtificial
Sequencea peptide derived from TOPK 75Arg Gly Leu Ser His Ser Pro
Trp Ala Val 1 5 10 7610PRTArtificial Sequencea peptide derived from
TOPK 76Ile Ile Leu Lys Val Ala Leu Asn Met Ala 1 5 10
7710PRTArtificial Sequencea peptide derived from TOPK 77Met Gln Lys
Leu Gly Phe Gly Thr Gly Val 1 5 10 7810PRTArtificial Sequencea
peptide derived from TOPK 78Ser Leu Cys Leu Ala Met Glu Tyr Gly Gly
1 5 10 7910PRTArtificial Sequencea peptide derived from TOPK 79Val
Ile Thr Asp Lys Ala Asp Ile Phe Ala 1 5 10 8010PRTArtificial
Sequencea peptide derived from TOPK 80Ala Leu Gly Thr Arg Pro Pro
Ile Asn Met 1 5 10 8110PRTArtificial Sequencea peptide derived from
TOPK 81Val Val Ile Lys Gly Asp Phe Glu Thr Ile 1 5 10
8210PRTArtificial Sequencea peptide derived from TOPK 82Ala Ile Ile
Leu Lys Val Ala Leu Asn Met 1 5 10 8310PRTArtificial Sequencea
peptide derived from TOPK 83Lys Val Ile Glu Leu Phe Ser Val Cys Thr
1 5 10 8410PRTArtificial Sequencea peptide derived from TOPK 84Glu
Thr Ile Lys Ile Cys Asp Val Gly Val 1 5 10 851899DNAHomo
sapiensCDS(202)..(1170) 85agcgcgcgac tttttgaaag ccaggagggt
tcgaattgca acggcagctg ccgggcgtat 60gtgttggtgc tagaggcagc tgcagggtct
cgctgggggc cgctcgggac caattttgaa 120gaggtacttg gccacgactt
attttcacct ccgacctttc cttccaggcg gtgagactct 180ggactgagag
tggctttcac a atg gaa ggg atc agt aat ttc aag aca cca 231 Met Glu
Gly Ile Ser Asn Phe Lys Thr Pro 1 5 10 agc aaa tta tca gaa aaa aag
aaa tct gta tta tgt tca act cca act 279Ser Lys Leu Ser Glu Lys Lys
Lys Ser Val Leu Cys Ser Thr Pro Thr 15 20 25 ata aat atc ccg gcc
tct ccg ttt atg cag aag ctt ggc ttt ggt act 327Ile Asn Ile Pro Ala
Ser Pro Phe Met Gln Lys Leu Gly Phe Gly Thr 30 35 40 ggg gta aat
gtg tac cta atg aaa aga tct cca aga ggt ttg tct cat 375Gly Val Asn
Val Tyr Leu Met Lys Arg Ser Pro Arg Gly Leu Ser His 45 50 55 tct
cct tgg gct gta aaa aag att aat cct ata tgt aat gat cat tat 423Ser
Pro Trp Ala Val Lys Lys Ile Asn Pro Ile Cys Asn Asp His Tyr 60 65
70 cga agt gtg tat caa aag aga cta atg gat gaa gct aag att ttg aaa
471Arg Ser Val Tyr Gln Lys Arg Leu Met Asp Glu Ala Lys Ile Leu Lys
75 80 85 90 agc ctt cat cat cca aac att gtt ggt tat cgt gct ttt act
gaa gcc 519Ser Leu His His Pro Asn Ile Val Gly Tyr Arg Ala Phe Thr
Glu Ala 95 100 105 aat gat ggc agt ctg tgt ctt gct atg gaa tat gga
ggt gaa aag tct 567Asn Asp Gly Ser Leu Cys Leu Ala Met Glu Tyr Gly
Gly Glu Lys Ser 110 115 120 cta aat gac tta ata gaa gaa cga tat aaa
gcc agc caa gat cct ttt 615Leu Asn Asp Leu Ile Glu Glu Arg Tyr Lys
Ala Ser Gln Asp Pro Phe 125 130 135 cca gca gcc ata att tta aaa gtt
gct ttg aat atg gca aga ggg tta 663Pro Ala Ala Ile Ile Leu Lys Val
Ala Leu Asn Met Ala Arg Gly Leu 140 145 150 aag tat ctg cac caa gaa
aag aaa ctg ctt cat gga gac ata aag tct 711Lys Tyr Leu His Gln Glu
Lys Lys Leu Leu His Gly Asp Ile Lys Ser 155 160 165 170 tca aat gtt
gta att aaa ggc gat ttt gaa aca att aaa atc tgt gat 759Ser Asn Val
Val Ile Lys Gly Asp Phe Glu Thr Ile Lys Ile Cys Asp 175 180 185 gta
gga gtc tct cta cca ctg gat gaa aat atg act gtg act gac cct 807Val
Gly Val Ser Leu Pro Leu Asp Glu Asn Met Thr Val Thr Asp Pro 190 195
200 gag gct tgt tac att ggc aca gag cca tgg aaa ccc aaa gaa gct gtg
855Glu Ala Cys Tyr Ile Gly Thr Glu Pro Trp Lys Pro Lys Glu Ala Val
205 210 215 gag gag aat ggt gtt att act gac aag gca gac ata ttt gcc
ttt ggc 903Glu Glu Asn Gly Val Ile Thr Asp Lys Ala Asp Ile Phe Ala
Phe Gly 220 225 230 ctt act ttg tgg gaa atg atg act tta tcg att cca
cac att aat ctt 951Leu Thr Leu Trp Glu Met Met Thr Leu Ser Ile Pro
His Ile Asn Leu 235 240 245 250 tca aat gat gat gat gat gaa gat aaa
act ttt gat gaa agt gat ttt 999Ser Asn Asp Asp Asp Asp Glu Asp Lys
Thr Phe Asp Glu Ser Asp Phe 255 260 265 gat gat gaa gca tac tat gca
gcg ttg gga act agg cca cct att aat 1047Asp Asp Glu Ala Tyr Tyr Ala
Ala Leu Gly Thr Arg Pro Pro Ile Asn 270 275 280 atg gaa gaa ctg gat
gaa tca tac cag aaa gta att gaa ctc ttc tct 1095Met Glu Glu Leu Asp
Glu Ser Tyr Gln Lys Val Ile Glu Leu Phe Ser 285 290 295 gta tgc act
aat gaa gac cct aaa gat cgt cct tct gct gca cac att 1143Val Cys Thr
Asn Glu Asp Pro Lys Asp Arg Pro Ser Ala Ala His Ile 300 305 310 gtt
gaa gct ctg gaa aca gat gtc tag tgatcatctc agctgaagtg 1190Val Glu
Ala Leu Glu Thr Asp Val 315 320 tggcttgcgt aaataactgt ttattccaaa
atatttacat agttactatc agtagttatt 1250agactctaaa attggcatat
ttgaggacca tagtttcttg ttaacatatg gataactatt 1310tctaatatga
aatatgctta tattggctat aagcacttgg aattgtactg ggttttctgt
1370aaagttttag aaactagcta cataagtact ttgatactgc tcatgctgac
ttaaaacact 1430agcagtaaaa cgctgtaaac tgtaacatta aattgaatga
ccattacttt tattaatgat 1490ctttcttaaa tattctatat tttaatggat
ctactgacat tagcactttg tacagtacaa 1550aataaagtct acatttgttt
aaaacactga accttttgct gatgtgttta tcaaatgata 1610actggaagct
gaggagaata tgcctcaaaa agagtagctc cttggatact tcagactctg
1670gttacagatt gtcttgatct cttggatctc ctcagatctt tggtttttgc
tttaatttat 1730taaatgtatt ttccatactg agtttaaaat ttattaattt
gtaccttaag catttcccag 1790ctgtgtaaaa acaataaaac tcaaatagga
tgataaagaa taaaggacac tttgggtacc 1850agaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 189986322PRTHomo sapiens 86Met Glu
Gly Ile Ser Asn Phe Lys Thr Pro Ser Lys Leu Ser Glu Lys 1 5 10 15
Lys Lys Ser Val Leu Cys Ser Thr Pro Thr Ile Asn Ile Pro Ala Ser 20
25 30 Pro Phe Met Gln Lys Leu Gly Phe Gly Thr Gly Val Asn Val Tyr
Leu 35 40 45 Met Lys Arg Ser Pro Arg Gly Leu Ser His Ser Pro Trp
Ala Val Lys 50 55 60 Lys Ile Asn Pro Ile Cys Asn Asp His Tyr Arg
Ser Val Tyr Gln Lys 65 70 75 80 Arg Leu Met Asp Glu Ala Lys Ile Leu
Lys Ser Leu His His Pro Asn 85 90 95 Ile Val Gly Tyr Arg Ala Phe
Thr Glu Ala Asn Asp Gly Ser Leu Cys 100 105 110 Leu Ala Met Glu Tyr
Gly Gly Glu Lys Ser Leu Asn Asp Leu Ile Glu 115 120 125 Glu Arg Tyr
Lys Ala Ser Gln Asp Pro Phe Pro Ala Ala Ile Ile Leu 130
135 140 Lys Val Ala Leu Asn Met Ala Arg Gly Leu Lys Tyr Leu His Gln
Glu 145 150 155 160 Lys Lys Leu Leu His Gly Asp Ile Lys Ser Ser Asn
Val Val Ile Lys 165 170 175 Gly Asp Phe Glu Thr Ile Lys Ile Cys Asp
Val Gly Val Ser Leu Pro 180 185 190 Leu Asp Glu Asn Met Thr Val Thr
Asp Pro Glu Ala Cys Tyr Ile Gly 195 200 205 Thr Glu Pro Trp Lys Pro
Lys Glu Ala Val Glu Glu Asn Gly Val Ile 210 215 220 Thr Asp Lys Ala
Asp Ile Phe Ala Phe Gly Leu Thr Leu Trp Glu Met 225 230 235 240 Met
Thr Leu Ser Ile Pro His Ile Asn Leu Ser Asn Asp Asp Asp Asp 245 250
255 Glu Asp Lys Thr Phe Asp Glu Ser Asp Phe Asp Asp Glu Ala Tyr Tyr
260 265 270 Ala Ala Leu Gly Thr Arg Pro Pro Ile Asn Met Glu Glu Leu
Asp Glu 275 280 285 Ser Tyr Gln Lys Val Ile Glu Leu Phe Ser Val Cys
Thr Asn Glu Asp 290 295 300 Pro Lys Asp Arg Pro Ser Ala Ala His Ile
Val Glu Ala Leu Glu Thr 305 310 315 320 Asp Val 8722DNAArtificial
Sequencea PCR primer for the TCR analysis 87gtctaccagg cattcgcttc
at 228824DNAArtificial Sequencea PCR primer for the TCR analysis
88tcagctggac cacagccgca gcgt 248921DNAArtificial Sequencea PCR
primer for the TCR analysis 89tcagaaatcc tttctcttga c
219024DNAArtificial Sequencea PCR primer for the TCR analysis
90ctagcctctg gaatcctttc tctt 24
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References